Waxholm Space Atlas of the Sprague Dawley Rat Brain

YNIMG-11256; No. of pages: 13; 4C: 3, 4, 5, 7, 8 NeuroImage xxx (2014) xxx–xxx

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NeuroImage

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1 Waxholm Space atlas of the Sprague Dawley rat brain

2Q1 Eszter A. Papp a,TrygveB.Leergaarda, Evan Calabrese b, G. Allan Johnson b, Jan G. Bjaalie a,⁎

3 a Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway 4 b Center for In Vivo Microscopy, Department of Radiology, Duke University Medical Center, Durham, NC, USA

5 article info abstract

6 Article history: Three-dimensional digital brain atlases represent an important new generation of neuroinformatics tools for 18 7 Accepted 1 April 2014 understanding complex brain anatomy, assigning location to experimental data, and planning of experiments. 19 8 Available online xxxx We have acquired a microscopic resolution isotropic MRI and DTI atlasing template for the Sprague Dawley rat 20 brain with 39 μm isotropic voxels for the MRI volume and 78 μm isotropic voxels for the DTI. Building on this 21 9 Keywords: template, we have delineated 76 major anatomical structures in the brain. Delineation criteria are provided for 22 10 Digital brain atlas 23 11 Waxholm Space each structure. We have applied a spatial reference system based on internal brain landmarks according to the 24 12 Sprague Dawley Waxholm Space standard, previously developed for the mouse brain, and furthermore connected this spatial 13 Rat brain template reference system to the widely used stereotaxic coordinate system by identifying cranial sutures and related 25 14 Segmentation stereotaxic landmarks in the template using contrast given by the active staining technique applied to the tissue. 26 15 Magnetic resonance imaging With the release of the present atlasing template and anatomical delineations, we provide a new tool for spatial 27 16 Diffusion tensor imaging orientation analysis of neuroanatomical location, and planning and guidance of experimental procedures in the 28 17 Neuroinformatics rat brain. The use of Waxholm Space and related infrastructures will connect the atlas to interoperable resources 29 and services for multilevel data integration and analysis across reference spaces. 30 31 © 2014 Published by Elsevier Inc. 323335 34 36 Introduction angles without loss of image quality. Further, in these digital image 58 volumes intersecting planes can be studied within the same brain. 59 37 Atlases of the brain are commonly used in neuroscience as frame- Atlases are built on top of templates by classifying image voxels into 60 38 works for spatial orientation, indexing neuroanatomical location different anatomical labels based on image contrast in the template or 61 39 of entities or events being studied, and planning and guidance of other data aligned to the template. 62 40 experimental procedures. Conventional brain atlases are based on inter- This approach has produced a series of volumetric atlasing templates 63 41 pretation of histological material stained to reveal cyto-, chemo-, or for the mouse (Aggarwal et al., 2009; Chuang et al., 2011; Johnson et al., 64 42 myeloarchitecture, and consist of series of plates containing drawings 2010; Kovacevic et al., 2005; Ma et al., 2005, 2008) and rat brain 65 43 of anatomical details as observed from well-deﬁned standard cutting (Johnson et al., 2012; Lu et al., 2010; Nie et al., 2013; Rumple et al., 66 44 planes. Several such brain atlases are widely used today in both 2013; Schwarz et al., 2006; Schweinhardt et al., 2003; Valdes- 67 45 primates and humans (Mai et al., 2007; Martin and Bowden, 2000; Hernandez et al., 2011; Veraart et al., 2011). In MRI/DTI datasets, cranial 68 46 Paxinos et al., 1999), and in smaller experimental animals including landmarks are not readily recognizable or not present (skull removed), 69 47 rodents (Franklin, 1997; Lein et al., 2007; Paxinos and Franklin, 2012; and thus coordinate systems based on in-brain landmarks have been 70 48 Paxinos and Watson, 1982, 2007; Swanson, 1992, 2004). In rodent proposed (Kovacevic et al., 2005). In an effort to establish standard 71 49 atlases, spatial reference is provided by the standard stereotaxic coordi- spatial reference deﬁned by internal landmarks in the rodent brain, 72 50 nate system based on cranial landmarks aiding precise localization for Waxholm Space (WHS, Hawrylycz et al., 2011) was introduced and 73 51 stereotaxic brain surgery (Paxinos and Watson, 1982). implemented initially in the mouse brain (Johnson et al., 2010). The 74 52 Recent progress in brain imaging has led to the introduction of a new highest resolution template published for the rat is from the Wistar 75 53 type of atlas based onUNCORRECTED volumetric template images of the brain strain and contains PROOF both MRI and DTI of the same brains (Johnson 76 54 (Aggarwal et al., 2011; Evans et al., 2012; Toga et al., 2006). While lim- et al., 2012). These templates are currently aligned to the stereotaxic 77 55 ited in resolution compared to microscopy, magnetic resonance imag- coordinate system of the Paxinos and Watson atlas. 78 56 ing (MRI) and diffusion tensor imaging (DTI) volumes acquired with For the Sprague Dawley strain, equally widely used as the Wistar, the 79 57 isotropic voxels allow the image to be resliced and viewed in arbitrary highest resolution MRI/DTI atlas was introduced by Veraart et al. (2011) 80 with 14 structures in selected regions delineated. A more completely 81 delineated whole brain atlas has been lacking. In the present study, 82 ⁎ Corresponding author at: Department of Anatomy, Institute of Basic Medical Sciences, 83 University of Oslo, P.O. Box 1105 Blindern, N-0317 Oslo, Norway. Fax: +47 22851278. we further investigate to which extent high resolution MRI data can E-mail address: [email protected] (J.G. Bjaalie). be used for detailed brain-wide delineation of a larger number of 84

85 structures. Employing ex vivo MRI data allowing detailed boundary resolution was 39 μm. Diffusion tensor datasets were acquired using a 145 86 delineation (Jiang and Johnson, 2010; Johnson et al., 2002, 2007, 2010, spin-echo pulse sequence (TR = 100 ms, TE = 16.2 ms, NEX = 1). Dif- 146 87 2012; Nieman et al., 2006; Veraart et al., 2011), we present a new fusion preparation was accomplished using a modified Stejskal–Tanner 147 88 volumetric atlas for the adult Sprague Dawley brain at a higher resolu- diffusion-encoding scheme 32 with a pair of unipolar, half-sine diffusion 148 89 tion than previously reported (MRI at 39 μm and DTI at 78 μmisotropic gradient waveforms (width δ = 4 ms, separation Δ = 8.5 ms, gradient 149 90 voxels). The atlas contains 76 anatomical regions along with delineation amplitude = 450 mT/m). One b0 image and six high b-value images 150 91 criteria for multiple types of image contrast. More detailed parcella- (b = 1500 s/mm2) were acquired with diffusion sensitization along 151 92 tions are currently ongoing for the hippocampus (L.J. Kjonigsen, T.B. each of six non-collinear diffusion gradient vectors: [1, 1, 0], [0, 1, 1], 152 93 Leergaard, M.P. Witter, and J.G. Bjaalie, in preparation; see also Bjaalie [1, 0, 1], [1, −1, 0], [0, 1, −1], and [−1, 0, 1]. The acquisition matrix 153 94 et al., 2013). In this template, we have implemented Waxholm Space was 512 × 256 × 256 over a 40 × 20 × 20 mm FOV. The Nyquist isotropic 154 95 in the rat brain for the first time, and documented its definition spatial resolution was 78 μm. All images were derived from fully 155 96 according to principles compatible with those in the mouse brain. sampled k-space data with no zero-filling. All images had a signal-to- 156

97 Further, we have connected the atlas to the stereotaxic coordinate noise-ratio (SNR) of 30 or greater. The long TR relative to T1 and the 157 98 system by identifying key cranial landmarks in the template. This long TE relative to T2, give the b0 image strong T2-weighting. We there- 158 99 creates a platform for interoperability across atlases and coordinate fore refer to the b0 image as T2-weighted below. The SNR for the aver- 159 100 systems in the rat brain. age isotropic DWI was ~120, and the SNR for the individual DWIs was 160

101 We envision and encourage this Waxholm Space atlas to be a com- ~62. The SNR for the T2*-weighted gradient echo was estimated ~50. 161 102 munity resource. The atlas and the underlying template are provided The DWI and anatomical images were acquired in a continuous session 162 103 open access, intended for reﬁnement and expansion. and were inherently co-registered. 163

104 Methods Image reconstruction and post-processing 164

105 Diffusion magnetic resonance images were acquired at microscopic After image reconstruction, all MRI volumes were registered to the 165

106 resolution from an adult male Sprague Dawley rat at the Duke Center standard T2-weighted b0 image using an Advanced Normalization 166 107 for in vivo microscopy. The brain was scanned ex vivo, residing intact Tools (http://www.picsl.upenn.edu/ANTS/) 9-parameter rigid afﬁne 167

108 in the cranium. T2 and T2*-weighted (gradient echo) anatomical images registration to correct for the linear component of eddy current distor- 168 109 and diffusion weighted images (DWI, Mukherjee et al., 2008), were ac- tions. Diffusion tensors were calculated at each voxel with multivariate 169 110 quired, reconstructed, and resampled to a common isotropic resolution linear ﬁtting using the TrackVis and Diffusion toolkit (http://trackvis. 170 111 of 39 μm. Image contrast in the anatomical MRI and diffusion tensor org). Finally, data were organized into consistent ﬁle architecture and 171 112 images was used to delineate 76 brain structures. All original images, archived in NIfTI format (http://nifti.nimh.nih.gov) in an onsite Oracle 172 113 the segmentation volume, and a hierarchical catalog of delineated struc- database. Fractional anisotropy (FA) values were computed from the 173 114 tures are shared through the INCF Software Center. three eigenvalues after tensor decomposition (Le Bihan et al., 2001). 174 All DWI images were resampled with no interpolation to match the 175

115 Animal preparation voxel size of the higher resolution T2*-weighted anatomical dataset 176 using the Matlab (MathWorks, Natick, MA) NIfTI Toolkit. This was 177 116 All animal procedures and experiments were approved by the Duke necessary for co-viewing the images as overlays during segmentation. 178 117 University Institutional Animal Care and Use Committee. An adult male An 8-bit binary mask was generated by thresholding the DWI volume 179 118 Sprague Dawley rat (age 80 days, weight 397.6 g, Charles River, using Matlab to hide non-brain structures such as the skull and soft 180 119 Wilmington, MA, USA) was actively stained with a mixture of formalin tissue. 181 120 and ProHance (Gadoteridol, Bracco Diagnostics, Inc., Princeton, NJ) to 121 increase MRI signal to noise ratio while preserving the tissue (Johnson Image segmentation 182 122 et al., 2002). The animal was anesthetized by intraperitoneal injection 123 of a mixture of nembutal (Ovation Pharmaceuticals, Inc., Lake Forest, Anatomical regions were delineated using ITK-SNAP software v2.2.0 183 124 IL) and butorphanol, and transcardially perfused with 0.9% saline and (Yushkevich et al., 2006; www.itksnap.org). The MRI data were viewed 184 125 ProHance (10:1 v:v) for 4 min followed by a ﬂush of ProHance in 10% in ITK-SNAP using the default 16-bit grayscale color map (black to 185 126 phosphate buffered formalin (1:10 v:v). The head with the brain in white), where dark areas in the image correspond to low signal intensi- 186 127 situ within the cranium was removed and stored in buffered formalin ty and bright areas correspond to high signal intensity. Neuroanatomi- 187 128 for at least 24 h. Tissue was rehydrated by immersion in a 1:200 solu- cal boundaries were delineated on basis of image contrast observed in 188

129 tion of ProHance/saline for 72 h. The head was trimmed to fit into an T2-weighted, T2*-weighted, and diffusion weighted images, including 189 130 acrylic sample holder that fits in the RF coil, and surrounded by fomblin, RGB maps of principal eigenvector orientation with fractional anisotro- 190 131 aperfluorocarbon that minimizes susceptibility artifacts at the interface. py represented by image intensity (DTI maps). Two standard rat brain 191 atlases were used as initial anatomical reference (Paxinos and Watson, 192 132 dMRI image acquisition 2007; Swanson, 2004). Additional anatomical criteria (see the Results 193 UNCORRECTEDsection) were employed PROOF to close anatomical boundaries when these 194 133 Microscopic MRI and DWI data were acquired at the Duke Center for were not unequivocally visible in the MRI or DTI images. The neuroana- 195 134 In Vivo Microscopy using a 7 T small animal MRI system (Magnex tomical nomenclature was adopted from the Paxinos and Watson 196 135 Scientific, Yarnton, Oxford, UK) equipped with 650 mT/m Resonance (2007) atlas of the rat brain. The left and right hemispheres of the 197 136 Research gradient coils (Resonance Research, Inc., Billerica, MA, USA), brain were delineated individually. At one location, a small unilateral 198 137 and controlled with a General Electric Signa console (GE Medical Sys- magnetic field interference artifact prevented delineation (see also the 199 138 tems, Milwaukee, WI). The specimen was imaged in a custom 30 mm Neuroanatomical delineations section). The boundary in question was 200Q3 139 diameter × 50 mm long solenoid RF coil fabricated from a continuous then manually transferred from the other side, taking into consideration 201 140 sheet of high-frequency microwave substrate (Roger Corp, Rogers, CT, aslightleft–right tilt of the coronal plane, making structures on the 202

141 USA). T2*-weighted gradient recalled echo (GRE) anatomic images right side of the brain appear 1–2 slices (39–78 μm) towards anterior 203 142 were acquired using a 3D sequence (TR = 50 ms, TE = 8.3 ms, NEX = compared to the left side. 204 143 2, α = 60°). The acquisition matrix was 1024 × 512 × 512 over a Semi-automatic segmentation was performed in adequate high- 205 144 40 × 20 × 20 mm ﬁeld of view (FOV). The Nyquist isotropic spatial contrast regions using the intensity region based SNaP algorithm 206

207 implemented in ITK-SNAP, producing a segmentation core for a limited T2 and T2*-weighted) and diffusion weighted MR images (Fig. 1). The 238 208 number of structures. Manual segmentation steps were taken iterative- structural images demonstrate architectural details in both gray and 239 209 ly for all delineated regions, performed by a single investigator based white matter with sufﬁcient contrast and spatial resolution to identify 240 210 on combined observations in the different dMRI maps in coronal, tracts, regions and nuclei. Diffusion weighted maps, FA maps and color 241 211 sagittal, and horizontal planes. Our delineation of boundaries was maps of principal diffusion orientations in the tissue reveal additional 242 212 aided by inspection of histological maps provided in the reference features useful for identifying and completing several anatomical 243

213 atlases (Paxinos and Watson, 2007; Swanson, 2004), as well as by series boundaries (Fig. 1). The delineations were primarily based on T2* and 244 214 of histological sections from brains of other Sprague Dawley specimens, DTI images, which allow identiﬁcation of most boundaries as reported 245 215 cut in coronal, sagittal, and horizontal planes, and stained for myelin below. 246 216 (Woelche, 1942) and cytoarchitecture (material from Leergaard et al., 217 2010; S. Lillehaug, J.G. Bjaalie, and T.B. Leergaard, unpublished results). Spatial reference system 247

218 Thus, changes in T2 and T2* signal intensity were compared to changes 219 in cell densities observed in thionine stained sections from correspond- We have applied Waxholm Space (WHS) on the atlasing template to 248 220 ing regions, while changes in DTI color maps were interpreted based provide standard spatial reference. WHS has been developed for the ro- 249 221 on observation of corresponding myelin stained fiber orientations. dent brain by the INCF Digital Atlasing Task Force (Hawrylycz et al., 250 222 Structures from the hippocampal and parahippocampal region, delin- 2011; Johnson et al., 2010), and is here implemented for the rat brain 251 223 eated in the same template, were taken from a parallel study (L.J. for the first time. Waxholm Space employs a continuous three- 252 224 Kjonigsen, T.B. Leergaard, M.P. Witter, and J.G. Bjaalie, in preparation). dimensional Cartesian coordinate system, with its origin set at the de- 253 cussation of the anterior commissure (Fig. 2). We identified the anterior 254

225 Data sharing commissure in the template based on its high contrast in the T2*andDTI 255 images, and we located the origin of WHS at the intersection of: a) the 256 226 Original high resolution anatomical MRI and diffusion weighted mid-sagittal plane, b) a coronal plane passing midway (rostro-caudal) 257 227 image volumes of the presented Waxholm Space template, together through the decussation of the anterior and posterior part of the anteri- 258 228 with anatomical segmentations and the corresponding label descrip- or commissure, and c) a horizontal plane passing midway through 259 229 tions are made available through the INCF Software Center (http:// the most dorsal and ventral aspect of the decussation of the anterior 260 230 software.incf.org/) in formats compatible with ITK-SNAP and the commissure (Fig. 2). This definition is compatible with the WHS 261 231 Mouse BIRN Atlasing Toolkit (MBAT, Lee et al., 2010), including struc- definition for the mouse brain (Johnson et al., 2010). In the present rat 262 232 ture hierarchy in XML (.ilf) format. Volumetric images are provided as brain template the WHS origin was identified at (NIfTI) coordinates 263 233 standard Neuroimaging Informatics Technology Initiative (NIfTI) files. 623 (coronal), 268 (sagittal), and 248 (horizontal). 264 The WHS coordinate system (Hawrylycz et al., 2011)isdefined in a 265 234 Results brain oriented in the flat skull position, where the height of cranial land- 266 marks lambda and bregma is at the same horizontal level (Paxinos and 267 235 Volumetric atlasing template Watson, 1982). Our MRI template was acquired approximating this 268 position. Although bony structures are generally invisible in MRI, it 269 236 We present an atlasing template for the Sprague Dawley rat brain was possible to observe the coronal, sagittal, and lambdoid skull sutures 270

237 consisting of high resolution, contrast enhanced structural (including in the T2*-weighted images due to diffusion of the contrast agent into 271

UNCORRECTED PROOF

Fig. 1. MRI and DTI contrasts used for anatomical delineations in the atlas: T2*-weighted (A, A′); diffusion weighted (DWI; B, B′); diffusion tensor (DTI, C,C′); fractional anisotropy (FA, D,D′). Features observed in the individual images were used in combination to delineate anatomical structures. Frame in A indicates the area enlarged in A′–D′. Yellow arrowheads indicate layer 2 of the piriform cortex, appearing as a continuous band in DWI (B′), but not in the other images. Red arrowheads indicate the boundary between the caudate–putamen

complex (striatum) and the globus pallidus, visible as a thin dark line in T2*images(A′). Insets in C and C′ show the RGB color codes for DTI orientation. aca, anterior part of the anterior commissure; AP, anteroposterior; BF, basal forebrain; Cx, cerebral cortex; DV, dorsoventral; EP, entopeduncular nucleus; ic, internal capsule; ML, mediolateral; Str, striatum; OB, olfactory bulb; rf, rhinal ﬁssure. Scale bars: 1 mm.

Fig. 2. The Waxholm Space coordinate system. The three axes (blue lines) and the coordinate system origin are identiﬁed in relation to the anterior commissure in sagittal (A), coronal (B),

and horizontal (C) T2*-weighted MRI slices. 3D rendering of the brain surface shows overall position of the origin within the brain (D). For coordinate system deﬁnition, see the Results section. aci, intrabulbar part of the anterior commissure; aca, anterior part of the anterior commissure; ac, anterior commissure; acp, posterior part of the anterior commissure. Scale bar: 1 mm.

272 the connective tissue. We could thus identify bregma at NIfTI coordi- differentiated from the cerebral cortex on basis of a distinct difference 303 273 nates 653 coronal; 266 sagittal; and 440 horizontal, and lambda at in FA value (high signal intensity) and principal diffusion orientation 304 274 NIfTI coordinates 442 coronal; 268 sagittal; and 464 horizontal. These in the DTI maps (Figs. 1, 4, and 5). The cingulum was not separately 305 275 coordinates allow the application of the widely used standard skull- delineated here, but is readily identified in DTI maps due to its 306 276 based stereotaxic coordinate system (Paxinos and Watson, 1982)to anterioposterior fiber orientation contrasting with the mediolaterally 307 277 the WHS template. We further also calculated that our spatial template oriented callosal fibers (Fig. 5C). The boundary of the external capsule 308 278 deviates 4° from the flat skull position (Fig. 3B). with the dorsal striatum was clearly seen on basis of gray and white 309

matter contrast visible in the T2-weighted and T2*-weighted images 310 279 Neuroanatomical delineations (Figs. 4D,G). The ventral part of the external capsule forms a thin 311 sheet conﬁning the striatum, and further posterior encompassing 312 280 For the ﬁrst release of the atlas, we have delineated 76 anatomical the hippocampal formation and the subiculum. The most ventral aspect 313

281 structures (Table 1) on basis of observations in the T2*-weighted and of this thin sheet is anteriorly best distinguished in DTI (mostly 314 282 diffusion weighted images in combination with relevant information dorsoventral orientation), while the posterior part is best seen in the 315

283 from published atlas resources (Paxinos and Watson, 2007; Swanson, T2*-weighted images (low signal intensity). 316 284 2004) and literature. The delineations cover the whole brain, including 285 the olfactory bulb and the ventricular system, as well as the spinal cord Anterior commissure 317 286 within 8.62 mm posterior to the skull. White matter bundles form The anterior commissure was delineated based on its high contrast 318

287 reliable landmarks that are clearly visible in both types of images and in both T2* and DTI maps (Figs. 1, 2). The structure was divided into 319 288 are readily distinguished from gray matter regions. Gray matter regions an intrabulbar part, located within the olfactory bulb, an anterior part 320 289 were thus partly deﬁned by surrounding white matter, and partly by including the decussation, and a posterior part comprising ﬁbers 321

290 more subtle differences in T2* and DTI contrast reﬂecting differences extending ventrolaterally from the anterior commissure between the 322 291 in average cellular density and/or cell diameter. The delineations were striatum and the basal forebrain. 323 292 manually corrected for a localUNCORRECTED magnetic ﬁeld interference artifact (likely PROOF 293 caused by bubbles of air or contrast agent trapped at the base of the Hippocampal white matter 324

294 brain) observed on the right side of the T2* image at the level of the The dorsal hippocampal commissure and its continuation in the 325 295 subthalamic region. The main criteria used to delineate white and gray alveus form a very thin layer with a thickness close to the voxel size 326

296 matter boundaries, emphasizing observations in T2* images and DTI employed (Fig. 5C). In the present version of the atlas, these structures 327 297 maps, are presented below. were not separately delineated along the dorsal aspect of the hippocam- 328 pus, but included as part of the corpus callosum and associated subcor- 329 298 White matter tical white matter. The alveus was, however, delineated as a separate 330 structure along the lateral and anterior aspects of the hippocampal for- 331 299 Corpus callosum and associated subcortical white matter mation, distinguished from the medially adjoining ﬁmbria by a slight 332

300 The corpus callosum, deep cerebral white matter, external capsule, difference in T2* signal intensity. The ventral hippocampal commissure 333 301 and cingulum constitute a continuous body of subcortical white mat- was delineated against the ﬁmbria based on its homogeneous appear- 334

302 ter and were delineated as one structure. These structures were ance in both DTI (mediolateral orientations, high FA) and T2*-weighted 335

Fig. 3. Stereotaxic skull landmarks identiﬁed in the MRI template. A–D show cranial sutures (arrowheads), including the coronal, sagittal and lambdoid sutures, in b0 images with inverted gray scale emphasizing the sutures. The position of the bregma (br) and lambda (la) landmarks is indicated. The WHS origin is marked with a white cross in panel B. E shows bregma and Q2 lambda as drawn by Paxinos and Watson (1998). F, 3D reconstruction of the lambdoid suture (meandering green line), matching the drawing shown in E. The lambda landmark is indicated by a green cross at the midpoint of the curve of best ﬁt along the suture (gray line). Scale bar: 5 mm.

336 images. The horns of the fimbria fill the space between the lateral ven- delineated separately due to its distinct orientation in the brain stem 351 337 tricles, the hippocampal formation, and the thalamus, as well as the highlighted in DTI maps. The internal capsule was identified by low 352

338 more anteriorly located septal region. The fimbria was delineated T2* signal intensity in the region located between the striatum and 353 339 against these structuresUNCORRECTED on basis of high FA values and characteristic globus pallidus laterally,PROOF and the thalamus medially (Figs. 4A,D,G). Dis- 354 340 diffusion orientations (Fig. 5). The boundary between the fimbria and persed fiber bundles penetrating the striatum were excluded from our 355

341 the stria terminalis was identified by the relatively lower T2* signal delineations. The ventral extent of the internal capsule was delineated 356 342 intensity in the fimbria. The fornix was distinguished by its distinct using DTI (slightly oblique dorsoventral fiber orientations). DTI maps 357

343 trajectory and its lower T2* intensity (Fig. 5A). The interpretation of were also used to delineate the anterior internal capsule against 358 344 DTI orientations in these regions was aided by inspection of correspond- the striatum. Otherwise, the descending ﬁber tracts were identiﬁed 359

345 ing myelin stained histological material. on the basis of low T2* values, high FA values and the distinct axial 360 diffusion orientations in these tracts. At several levels in the brain 361 346 Corticofugal pathways stem, the descending pyramidal tract was impossible to differentiate 362 347 The corticofugal ﬁber tracts (Figs. 1, 4–6), including the internal from the adjacent medial lemniscus. Here the dorsal boundary of the 363 348 capsule, cerebral peduncle, longitudinal fasciculus of the pons, and the pyramidal tract was extrapolated to the expected geometrical shape 364 349 pyramidal tract, were delineated as one continuous structure from the and position relative to the brain surface and to the midline of the 365 350 internal capsule to the level of the pyramidal decussation, which was brain. 366

Please cite this article as: Papp, E.A., et al., Waxholm Space atlas of the Sprague Dawley rat brain, NeuroImage (2014), http://dx.doi.org/10.1016/ j.neuroimage.2014.04.001 6 E.A. Papp et al. / NeuroImage xxx (2014) xxx–xxx t1:1 Table 1 Table 1 (continued) t1:2 Alphabetical list of white and gray matter structures included in the atlas with reference to Other structures t1:79 t1:3 chapters in the Results section. Central canal 3.7 t1:80 t1:4 White matter 3.4 Spinal cord 3.7 t1:81 t1:5 Alveus of the hippocampus 3.4.3 Ventricular system 3.6 t1:82 t1:6 Anterior commissure, anterior part 3.4.2 t1:7 Anterior commissure, intrabulbar part 3.4.2 t1:8 Anterior commissure, posterior part 3.4.2 t1:9 Ascending fibers of the facial nerve 3.4.8 Medial lemniscus 367 t1:10 Brachium of the superior colliculus 3.4.11 368 t1:11 Commissural stria terminalis 3.4.6 The medial lemniscus was clearly visible with low signal intensity in t1:12 Commissure of the inferior colliculus 3.4.11 the T2* weighted images from the level of the substantia nigra through 369 t1:13 Commissure of the superior colliculus 3.4.11 the tegmentum to its entry into the thalamus (Fig. 5). At other levels 370 t1:14 Corpus callosum and associated subcortical white matter 3.4.1 of the brain stem it was hard to distinguish and thus not delineated in 371 t1:15 Corticofugal pathways 3.4.4 372 t1:16 Facial nerve 3.4.8 the present version of the atlas. The decussation of the medial lemnis- t1:17 Fasciculus retroflexus 3.4.6 cus, however, was delineated by its dorsoventral fiber orientation, and 373 t1:18 Fimbria of the hippocampus 3.4.3 separated from the pyramidal decussation by its lower FA values and 374 t1:19 Fornix 3.4.3 slightly lower T2* signal intensity. 375 t1:20 Genu of the facial nerve 3.4.8 t1:21 Habenular commissure 3.4.6 376 t1:22 Inferior cerebellar peduncle 3.4.12 Thalamic tracts t1:23 Mammillothalamic tract 3.4.6 Several distinct white matter tracts associated with the thalamus 377 t1:24 Medial lemniscus 3.4.5 were clearly visible and delineated on basis of T2*-weighted images 378 t1:25 Medial lemniscus decussation 3.4.5 and DTI maps (Fig. 5; see, also Figs. 6F–H). The following tracts were 379 t1:26 Middle cerebellar peduncle 3.4.12 380 t1:27 Optic nerve 3.4.9 delineated and subsequently used to aid the delineation of the thalamic t1:28 Optic tract and optic chiasm 3.4.9 gray matter: 1) the mammillothalamic tract, 2) the fasciculus 381 t1:29 Posterior commissure 3.4.7 retroflexus (of Meynert), providing a prominent white matter landmark 382 t1:30 Pyramidal decussation 3.4.4 at the diencephalic–mesencephalic boundary, 3) the stria medullaris of 383 t1:31 Spinal trigeminal tract 3.4.10 the thalamus, 4) the habenular commissure, 5) the arc of the stria 384 t1:32 Stria medullaris of the thalamus 3.4.6 385 t1:33 Stria terminalis 3.4.6 terminalis (Gloor, 1955; Morgane et al., 2005), and 6) the commissural t1:34 Supraoptic decussation 3.4.9 stria terminalis. At the levels where the stria terminalis adjoins the bed 386 t1:35 Transverse fibers of the pons 3.4.12 nucleus of the stria terminalis, the two structures were distinguished 387 t1:36 Ventral hippocampal commissure 3.4.3 based on the FA maps. The posterior part of the stria terminalis is 388 t1:37 t1:38 Gray matter 3.5 intercalated between the internal capsule and the ventral fimbria, dis- 389 fi 390 t1:39 Basal forebrain region 3.5.7 tinguished by higher T2* values and a difference in ber orientations t1:40 Bed nucleus of the stria terminalis 3.5.10 (Fig. 5). 391 t1:41 Brain stem 3.5.23 t1:42 Caudal entorhinal field 3.5.2 Posterior commissure 392 t1:43 Cingulate cortex, area 2 3.5.2 The posterior commissure, forming a roof over the third ventricle at 393 t1:44 Deeper cerebellum 3.5.19 t1:45 Deeper layers of the superior colliculus 3.5.14 the boundary between the diencephalon and telencephalon, was delin- 394 t1:46 Dorsal-intermediate entorhinal area 3.5.2 eated based on its low signal intensity in T2*-weighted images. 395 t1:47 Dorsal-lateral entorhinal area 3.5.2 t1:48 Entopeduncular nucleus 3.5.5 Facial nerve 396 t1:49 Frontal association cortex 3.5.2 fi 397 t1:50 Globus pallidus 3.5.4 The facial nerve (7th cranial nerve, Fig. 6G) and its ascending bers t1:51 Glomerular layer of the accessory olfactory bulb 3.5.1 were delineated based on FA maps, whereas the genu of the facial 398 t1:52 Glomerular layer of the olfactory bulb 3.5.1 nerve was identified by its low intensity in T2*-weighted images. 399 t1:53 Hippocampal formation 3.5.2 t1:54 Hypothalamic region 3.5.12 fi 400 t1:55 Inferior colliculus 3.5.14 Optic ber system and supraoptic decussation t1:56 Inferior olive 3.5.20 The optic nerve was delineated based on its low intensity in T2*- 401 t1:57 Interpeduncular nucleus 3.5.16 weighted images, aided by FA maps, from its origin in the retina to the 402 t1:58 Medial entorhinal field 3.5.2 level where the right and left optic nerves merge in the optic chiasm. 403 t1:59 Molecular cell layer of the cerebellum 3.5.19 The optic chiasm and tract were delineated together as one structure 404 t1:60 Neocortex 3.5.2 405 t1:61 Nucleus of the stria medullaris 3.5.11 on basis of high FA values and DTI orientations. The optic tract was read- t1:62 Olfactory bulb 3.5.1 ily followed to the point where it adjoins the descending peduncle and 406 t1:63 Periaqueductal gray 3.5.17 curves laterally around the thalamus. The supraoptic decussation was 407 t1:64 Perirhinal cortex 3.5.2 distinguished from the optic tract and corticofugal pathways on the 408 t1:65 Periventricular grayUNCORRECTED 3.5.22 PROOF 409 t1:66 Pineal gland 3.5.13 basis of high T2*-signal intensity (Fig. 5A), lower FA values and slightly t1:67 Pontine nuclei 3.5.18 different DTI fiber orientations (Fig. 5C). 410 t1:68 Postrhinal cortex 3.5.2 t1:69 Pretectal region 3.5.14 Trigeminal nerve and spinal trigeminal tract 411 t1:70 Septal region 3.5.8 The sensory and motor roots of the trigeminal nerve and the spinal 412 t1:71 Spinal trigeminal nucleus 3.5.21 413 t1:72 Striatum 3.5.3 trigeminal tract were delineated together, readily distinguished by t1:73 Subiculum 3.5.2 high FA values and distinct low T2* signal intensity. Diffusion orienta- 414 t1:74 Substantia nigra 3.5.15 tions were used to differentiate the trigeminal nerve from the middle 415 t1:75 Subthalamic nucleus 3.5.6 cerebellar peduncle, and T2* contrast was used to delineate the spinal 416 t1:76 Superficial gray layer of the superior colliculus 3.5.14 417 t1:77 Thalamus 3.5.9 trigeminal tract against the medially located spinal trigeminal nucleus. t1:78 Ventral-intermediate entorhinal area 3.5.2 The spinal trigeminal tract was further differentiated from more 418 externally located white matter tracts (inferior cerebellar peduncle, 419

420 trapezoid body, vestibulocochlear nerve) on basis of its distinct axial dif- Gray matter 445 421 fusion orientations (Figs. 4G–I). Olfactory system 446 Within the olfactory and accessory olfactory bulb, the outer (glomer- 447 422 White matter of the tectum ular) layers were separately delineated based on the substantially lower 448

423 The commissures of the superior and inferior colliculi and the T2* signal intensity in these layers relative to the deeper layers. The 449 424 brachium of the superior colliculus were delineated on basis of distinct deeper layers of the olfactory and accessory olfactory bulb, the lateral 450

425 low T2* signal intensity, aided by high FA values and mediolateral DTI olfactory tract, and the anterior parts of the piriform cortex were delin- 451 426 orientations. eated as one structure (Figs. 1A, A′,B,B′), whereas the intrabulbar part of 452 the anterior commissure was delineated separately (see the Anterior 453 commissure section, above). Posterior to the olfactory bulb, the olfacto- 454 427 Cerebellar and precerebellar white matter ry system protrudes ventrally below the ventral pallidum and the orbit- 455 428 The white matter of the cerebellum and the layered organization of al and insular cortex. Anteriorly, the olfactory system is divided from the 456

429 the cerebellum was seen in both T2*andDTImaps(Figs. 4G–I). Howev- cerebral cortex by the rhinal incisure and the rhinal fissure. In more pos- 457 430 er, since the low intensity T2* signals of the cerebellar white matter were terior regions, where the rhinal fissure is shallow, the dorsal boundary 458 431 difficult to distinguish from the overlying granule cell layer (with medi- of the olfactory system was delineated on basis of the lower T2*signal 459 432 um intensity T2* signals) at several locations, we chose not to delineate intensity observed in the ventral and lateral orbital cortex, and insular 460 433 the cerebellar white matter separately (see also the Cerebellum sec- cortex, and further by the homogenous high T2* intensity seen in the ac- 461 434 tion). In the present version of the atlas the superior cerebellar peduncle cumbens nucleus. The piriform cortex, forming the most dorsolateral 462 435 was not delineated separately but included in the brain stem region. The part of the olfactory system, was identified as a homogeneous region 463

436 middle and inferior cerebellar peduncles were delineated using primar- appearing as relatively bright in T2* images, and with its distinctly curv- 464 437 ily DTI maps, aided by the T2*-weighted images. The delineations were ing layer 2 clearly visible in diffusion weighted images (Figs. 1A,B). The 465 438 truncated at the level where the ﬁber tracts enter the cerebellum, arbi- medial and ventromedial boundary of the olfactory system was deﬁned 466

439 trarily defined by a line connecting the lateral corner of the 4th ventricle against the navicular nucleus of the basal forebrain (with lower T2* 467 440 with the lateral recess of the 4th ventricle. The transverse fibers of the intensity) and the ventral pallidum (with distinct anteroposterior fiber 468

441 pons were delineated on the basis of low T2* and high FA values and orientations). The olfactory tubercle, located medial to the lateral 469 442 identiﬁed as a continuous white matter layer covering the external olfactory tract, could not be distinguished from the ventral pallidum 470 443 surface of the pontine nuclei. The transition to the middle cerebellar and was therefore not included. Towards posterior, the delineation of 471 444 peduncle was set where the ﬁbers leave the surface of pontine nuclei. the olfactory system was arbitrarily truncated at the anteroposterior 472

UNCORRECTED PROOF

Fig. 4. Overview of anatomical features and delineations. Atlas delineations are shown in coronal, horizontal and sagittal views (B,E,H), together with corresponding T2*(A,D,G) and DT (C,F,I) images showing corresponding and complementary features. Arrows indicate the positions of the different image planes. Insets in C, F, and I show the RGB color code used for DTI orientation. BF, basal forebrain region; BS, brain stem; Cb, cerebellum; Cx, cerebral cortex; ec, external capsule; EP, entopeduncular nucleus; ﬁ, ﬁmbria; GP, globus pallidus; HC, hippocampal formation; HT, hypothalamic region; IC, inferior colliculus; ic, internal capsule; icp, inferior cerebellar peduncle; OB, olfactory bulb; opt, optic tract; PAG, periaqueductal gray; PRh, perirhinal cortex; S, subiculum; SC, superior colliculus; SN, substantia nigra; SNu, septal nuclei; Sp5, spinal trigeminal nucleus; sp5, spinal trigeminal tract; Str, striatum; Thal, thalamus. Scale bars, 1 mm.

Fig. 5. Structural details in white and gray matter visible in corresponding T2* (A) and DTI (C) images, and with atlas delineations superimposed on the T2* image (B), from a coronal MRI slice through the hippocampus, thalamus, and hypothalamic region. 3 V, third ventricle; alv, alveus; BS, brain stem region; cc, corpus callossum; cg, cingulum; Cx, cerebral cortex; dhc, dorsal hippocampal commissure; ec, external capsule; f, fornix; fi, fimbria; fr, fasciculus retroflexus; HC, hippocampal formation; HT, hypothalamic region; ic, internal capsule; ml, medial lemniscus; mt, mammillothalamic tract; opt, optic tract; sm, stria medullaris; sox, supraoptic decussation; st, stria terminalis; Str, striatum; Thal, thalamus. Scale bar, 1 mm.

473 level where the rhinal ﬁssure becomes a shallow groove on the lateral same MRI/DTI material (L.J. Kjonigsen, T.B. Leergaard, M.P. Witter, and 486 474 surface of the cerebral cortex. J.G. Bjaalie, in preparation) based on criteria provided by Kjonigsen 487 et al. (2011). The structures included were the entorhinal, perirhinal 488 475 Cerebral cortex including the neocortex and the hippocampus and postrhinal cortices of the parahippocampal region, the subiculum, 489 476 The label neocortex in the present atlas comprises all parts of the ce- and the remaining hippocampal formation (cornu ammonis regions 490 477 rebral cortex not included in the olfactory system or in the hippocampal 1–3, the fasciola cinereum, and the dentate gyrus, together labeled as 491 478 and parahippocampal regions (Figs. 4–6). Only the frontal association hippocampal formation). 492 479 cortex and area 2 of the cingulate cortex were delineated as separate 480 structures. The frontal association cortex was delineated based on slight Striatum 493

481 differences in T2* contrast towards the orbital cortex and the secondary The striatum, comprising the caudate–putamen complex and the 494 482 motor cortex, whereas area 2 of the cingulate cortex was outlined with core and shell of the accumbens nucleus (Gerfen, 2004), was distin- 495

483 use of white matter landmarks in the T2*-weighted images. Delineations guished by its distinct low FA signal intensity, in addition to contrast 496 484 of structures in the hippocampal and parahippocampal regions were provided by surrounding structures (Figs.1,4D–I). The striatum is dor- 497 485 taken from a parallel investigation of hippocampal boundaries in the sally covered by white matter (the corpus callosum and external 498

UNCORRECTED PROOF

Fig. 6. Surface models of the Waxholm rat brain atlas showing the 3D shape and position of selected structures. 7n, facial nerve; ac, anterior commissure; aca, anterior commissure, anterior part; aci, anterior commissure, intrabulbar part; acp, anterior commissure, posterior part; BS, brain stem; Cb, cerebellum; cc, corpus callosum; Cx, cerebral cortex; DpG, deep gray layer of the superior colliculus; ec, external capsule; fi, fimbria; fr, fasciculus retroflexus; GP, globus pallidus; HC, hippocampal formation; HT, hypothalamic region; IC, inferior colliculus; ic, internal capsule; OB, olfactory bulb; PAG, periaqueductal gray matter; PRh, perirhinal cortex; PVG, periventricular gray matter; py, pyramidal tract; S, subiculum; SC, superior colliculus; SuG, superficial gray layer of the superior colliculus; sm, stria medullaris; SNu, septal nuclei; st, stria terminalis; Str, striatum; Thal, thalamus.

499 capsule) with low T2* intensity and high anisotropy (Fig. 4). The anterior the subthalamic nucleus appears with bright signal, while in the FA 562 500 protrusion of the accumbens nucleus was delineated based on its low image it appears as a dark region. As the T2* image shows signiﬁcant dis- 563 501 anisotropy and homogenous bright appearance in T2* images. The tortions in this region on the right side, the other two image modalities 564 502 medial boundary of the anterior striatum is given mostly by the lateral were used for the delineation. 565

503 ventricles (black/white in T2*-weighted images), in addition to the na- 504 vicular nucleus and anterior olfactory nucleus (with anteroposterior Basal forebrain region 566

505 DTI orientations) and the septal nuclei (with slightly lower T2* intensity, The basal forebrain is a large complex of smaller regions located 567 506 and relatively higher anisotropy). Further posterior, the internal capsule ventral to the striatum (Figs. 1, 4G,H, 5). In the current version of the 568

507 (characterized by low T2* intensity, and high anisotropy) and the globus atlas, the basal forebrain was provisionally defined as including brain 569 508 pallidus provide the medial/ventromedial boundary of the striatum. The regions located at the floor of the brain, between the olfactory system 570 509 anterior part of the striatum was delineated ventrally against the medial and hypothalamic region, inferior to the striatum and globus pallidus. 571 510 forebrain bundle and the ventral pallidum, both included in the basal For identification of boundaries, we refer to the delineation criteria for 572 511 forebrain region. The white matter of the medial forebrain bundle was the surrounding structures (the olfactory bulb, the cerebellar cortex, 573

512 easily identified by its low T2* signal intensity and high anisotropy the striatum and globus pallidus, the septum, the ventricular system, 574 513 (anteroposterior fiber orientations), while the rest of the ventral and the thalamus, and the hypothalamic region). 575 514 pallidum provides a boundary by its relatively higher anisotropy 515 compared to the striatum, showing mostly anteroposterior diffusion Septal region 576 516 orientations. Further posterior, the posterior limb of the anterior com- Although the septal nuclei are often considered part of the basal 577 517 missure (with distinct mediolateral fiber orientations) and the intersti- forebrain (http://neurolex.org/), we have here delineated the septal re- 578 518 tial nucleus of the posterior limb of the anterior commissure (with gion individually, defined as the region situated medially between the 579

519 relatively low T2* intensity) give the ventral boundary of the striatum. lateral ventricles, directly ventral to the corpus callosum, encompassing 580 520 The lateral/ventrolateral extent of the striatum, which is not delimited the anterior fornix, and separating the anterior hippocampal structures 581 521 by the external capsule, was delineated against the claustrum and the (the ﬁmbria and the ventral hippocampal commissure) from the dorsal 582

522 endopiriform nuclei (with darker T2* signals), and more posteriorly by peduncular cortex. The septal region was distinguished from surround- 583 523 the amygdaloid nuclei (with slightly darker, homogenous T2*contrast, ing areas by its bright T2* contrast, low anisotropy, and distinctive blend 584 524 lacking the typical striated appearance of the striatum). of dorsoventral and anteroposterior tissue orientations. The ventral 585 boundary of the septal region against the basal forebrain was approxi- 586 525 Globus pallidus mated based on the same characteristics, aided by a slight shift towards 587

526 The globus pallidus is readily distinguished medially from the white brighter T2* signal in the basal forebrain. 588 527 matter of the internal capsule by its bright T2* contrast. The dorsolateral 528 boundary towards the striatum is more difficult to see in the T2* images Thalamus 589 529 since the overall appearance of the tissue is striated in both regions. The For the present version of the atlas, we have delineated the thalamus 590 530 boundary is indicated by a thin band of slightly darker signal between as a single region without subdivisions, incorporating the main nuclear 591 531 the globus pallidus and the striatum (Figs. 1A′–D′; 4D,G), coinciding groups of the epithalamus, dorsal thalamus, and ventral thalamus 592 532 with a relatively sharp boundary given by higher FA values in the globus (Jones, 2007). The delineation was aided by the identification of distinct 593 533 pallidus (Figs. 4F,I), corresponding to the higher density of fibers in this white matter bundles surrounding and entering the thalamus (Figs. 6F– 594 534 region. The anterior part of the GP is ventrally delimited partly by the H; see the Subthalamic nucleus section, above). The overall shape of 595

535 posterior limb of the anterior commissure (with dark T2* signal, the thalamus is most apparent in horizontal view (Figs. 4D–F). The 596 536 mediolateral ﬁber orientation) and the associated interstitial nucleus round anterior surface of the thalamus is delimited towards lateral by 597

537 (with slightly brighter T2* signal), and partly by the ventral pallidum. the 3rd ventricle (with very bright T2* signal), the fornix, the stria 598 538 The distinct appearance of the globus pallidus with high densities of medullaris, the bed nucleus of the stria terminalis, and the arc of the 599 539 ﬁber bundles was used to delineate its ventral boundary against stria terminalis, and further lateral by the adjacent convex surface of 600 540 the ventral pallidum (included in the basal forebrain region) and the the internal capsule and the ﬁmbria of the hippocampus (white matter 601

541 sublenticular extended amygdala, aided also by slightly darker T2* with darker T2* contrast), and the bed nucleus (with slightly brighter 602 542 signal in the extended amygdala. The basal nucleus (of Meynert), inter- signal compared to the thalamus). The dorsal surface of the thalamus 603 543 calated between the globus pallidus and extended amygdala, was not is bounded by the 3rd ventricle and the stria medullaris of the hippo- 604 544 possible to identify separately and was therefore included in the delin- campus (Fig. 5). The lateral boundary of the anterior thalamus is given 605 545 eation of the globus pallidus. Further posterior, the globus pallidus is by the internal capsule, the fimbria, and the arc of the stria terminalis in- 606 546 ventrally adjacent to the amygdaloid nuclei showing darker FA signals. tercalated between the two other white matter bundles. The posterior 607 547 The posterior border of the globus pallidus is given by the inferosuperior boundary of the thalamus is defined by the optic tract, the hippocam- 608 548 segment of the stria terminalis (which is identified by high FA values) pus, and the ventricular system. The ventral boundary of the anterior 609 549 directly posterior to the internal capsule and anteroventral to the thalamus against the basal forebrain region was partly defined by the 610 550 fimbria. curvatures of the stria medullaris and fornix, and partly by the medial 611 UNCORRECTEDdivision of the bedPROOF nucleus of the stria terminalis, which appeared as 612 551 Entopeduncular nucleus azoneofbrightT2* contrast surrounding the fornix (Fig. 5). The 613 552 The entopeduncular nucleus, a small gray matter region embedded paraventricular hypothalamic nucleus, an area of dark T2* contrast 614 553 in the internal capsule, is clearly visible as an island with bright T2* tightly surrounding the ventral half of the 3rd ventricle, also contributes 615 554 signal and reticular appearance surrounded by white matter (Figs. 4A, to this boundary. Further posteriorly, the ventrolateral boundary of the 616

555 G). It is to some extent also visible as small fragments with reduced thalamus was identiﬁed using the noticeable dark shift in T2* contrast 617 556 fractional anisotropy in the FA maps. towards the basal forebrain in an arc extending the medial surface of 618 the internal capsule in the ventromedial direction (Fig. 5). The ventral 619 557 Subthalamic nucleus boundary of the anterior part of the thalamus towards the hypothalamic 620

558 The subthalamic nucleus is visible as a distinct island of bright T2* region was approximated by a curve following a zone with a slight drop 621 559 signal located dorsomedial to the cerebral peduncle, anterior to the in FA signal. From the level where the bed nucleus of the stria terminalis 622 560 substantia nigra, and ventral to the thalamus/zona incerta. The nucleus disappears and the medial magnocellular nuclei become visible medial- 623

561 was delineated using both T2*, T2, and FA images. In T2 and T2*images, ly (elongated structures running obliquely dorsolateral from the 3rd 624

625 ventricle towards posterior, with dark T2* signal), the zona incerta was Hypothalamic region 689 626 used to define the ventral boundary of the thalamus towards the basal The hypothalamus contains several smaller nuclei, some of which 690 627 forebrain region and the thalamus. can be recognized in the image material (e.g. the paraventricular and 691 628 The zona incerta can be considered as a part of the ventral thalamus the periventricular nuclei, and the pars compacta of the dorsomedial 692 629 (Jones, 2007) and is here thus included as part of the thalamus. This re- hypothalamic nucleus). These regions are not separately delineated in 693 630 gion is intercalated between the white matter bundles of the internal the present version of the atlas. The hypothalamic region is character- 694 631 capsule, the medial lemniscus, and the mammillothalamic tract, clearly ized by low anisotropy compared to the surrounding regions (Fig. 5). 695 632 defined by distinct mediolateral fiber orientations standing out against Its dorsal and ventral extents are mostly given by the thalamus, the 696 633 the oblique fiber orientations of the more laterally located internal brain stem, and the ventral brain surface. The optic chiasm and optic 697 634 capsule, and the low anisotropic values of the ventrally and medially tract, as well as the supraoptic decussation (all with very bright FA 698 635 located hypothalamic region (Fig. 5). Medially, between the zona signal) also contribute to the ventral and ventrolateral boundary. The 699 636 incerta on either side, at level of the reuniens thalamic nucleus and lateral dimensions of the hypothalamic region, where not defined by 700 637 reuniens area, the ventral extent of the thalamus was approximated the outer brain surface, were approximated against the slightly darker 701

638 on the basis of a reduction in fractional anisotropy towards the hypotha- T2* signal and higher anisotropy of the basal forebrain region. The 702 639 lamic region. The posterior boundary of the thalamus was delineated more posteriorly located peduncular part of the lateral hypothalamus 703 640 against several mesencephalic structures. The pretectal nuclei appear is here not identiﬁed separately, but rather included in the basal 704

641 dorsally as regions with slightly brighter T2* contrast and mediolateral forebrain region. The fornix and the surrounding bed nucleus of the 705 642 tissue orientation, giving a round lateral surface against the horns of stria terminalis contribute further to this lateral boundary. Similarly, 706 643 the thalamus, and a rather ﬂat surface towards the dorsal half of the ﬂat- the anterior part of the hypothalamus was estimated as a zone of bright 707

644 tened concave part of the thalamic crescent. This boundary is best seen T2* contrast located between the optic chiasm, the medial division of the 708 645 and thus approximated in horizontal view. At the dorsoventral midline bed nucleus of the stria terminalis, and the preoptic area (included in 709 646 of the posterior surface of the thalamus, it meets the periaqueductal the basal forebrain region). Directly posterior to the level where the 710

647 gray (with brighter T2* signals), at the anterioposterior level where mammillothalamic tract is located between the thalamus and the hypo- 711 648 the dorsal and ventral parts of the 3rd ventricle join. The fasciculus thalamus, the dorsal/dorsolateral border of the hypothalamic region is 712

649 retroﬂexus (with very dark T2* signals, and inferosuperior DTI orienta- given by the protruding reticular formation (characterized by darker 713 650 tions) and the surrounding parafascicular nucleus (which has brighter T2* signal with characteristic reticular pattern, here delineated as part 714 651 T2* signals compared to the thalamus, and slightly darker T2* signals of the brain stem) and medially by the periaqueductal gray. Due to the 715 652 than the periaqueductal gray matter) also contribute to this boundary lack of contrast between the posterior hypothalamic area and the 716 653 up to the posterior level where the periaqueductal gray appears medial- periaqueductal gray, this boundary was set to follow the ventral surface 717 654 ly between the right and left retroﬂex fasciculus. The ventral half of of the mammillothalamic tract. The most posterior part of the hypotha- 718

655 the crescent is delimited by the tectal part of the reticular formation lamic region is formed by the mammillary nuclei (with brighter T2* 719 656 (included in the brain stem), showing considerably darker T2* contrast contrast). 720 657 and characteristic reticular tissue structure picked up in the MRI. It 658 should be noted that the right side of the MR image shows severe distor- Pineal gland 721 659 tions at the posterior parts of the thalamus, and therefore the posterior Anterolaterally, the dorsal half of the pineal gland is adjacent to the 722 660 boundary on this side was largely approximated to be symmetrical to cortex, separated by a clear boundary and distinguished also by its 723

661 the left side. brighter T2* contrast. Otherwise the boundaries of the pineal gland are 724 readily given by the saturated or zero signal (depending on the presence 725 662 Bed nucleus of the stria terminalis of contrast agent) in the surrounding cerebrospinal ﬂuid. 726 663 The bed nucleus of the stria terminalis was identiﬁed as a zone of

664 relatively bright T2* signals located posterior of striatum, between the Tectum 727 665 anterior commissure, septal nuclei, internal capsule and the lateral The tectum was parcellated into the superﬁcial and deep layers of 728 666 ventricle. The lateral and medial boundaries of the anterior part of the the superior colliculus, the inferior colliculus and the commissure of 729 667 region are given by the internal capsule and the septum, respectively. the inferior colliculus. The region between the thalamus and superior 730 668 Posterior of the level where the 3rd ventricle merges with the lateral colliculus (containing several pretectal nuclei) was delineated as the 731 669 ventricles, the bed nucleus of the stria terminalis is medially delimited pretectal region. The superﬁcial gray layer of the superior colliculus 732

670 by the anterior thalamus (paratenial nucleus, with slightly darker T2* was readily identiﬁed as a zone of bright T2* contrast located between 733 671 signal), the stria medullaris (with high FA and dorsoventral DTI orienta- the brain surface and the deeper layers of the superior colliculus. The 734 672 tions), and the fornix (with oblique anteroposterior and dorsoventral deeper layers of the superior colliculus (including the optical nerve 735 673 diffusion orientations). Its dorsomedial boundary is given by the lateral layer, and the intermediate and deep gray and white layers) were 736 674 ventricle, whereas its dorsolateral boundary is adjacent to the stria here collectively delineated. The posterior boundary of the superior 737 675 terminalis (with bright FA signals). Complementing the arc formed by colliculus was set at the level of the groove separating the superior 738 676 the stria terminalis and the dorsal part of its bed nucleus, the ventral gray layer from the inferior colliculus, and given by the homogeneous 739 677 740 extension of the bed nucleusUNCORRECTED of the stria terminalis encompasses the T2* appearance ofPROOF the inferior colliculus and the commissure of the 678 anterior side of the fornix, appearing as a curved zone of bright T2*signal inferior colliculus. The anterior boundary of the superior colliculus 741 679 surrounding the fornix. The bed nucleus of the stria terminalis can be was more difﬁcult to distinguish from the pretectal nuclei, but was 742 680 followed towards posterior along the fornix to the level where the approximated by observing zones with subtle alternating differences 743

681 fornix becomes detached from the thalamic surface. in T2* contrast, probably reﬂecting the different layers of the superior 744 colliculus. The ventral boundary of the superior colliculus against the 745 682 Nucleus of the stria medullaris reticular formation was delineated using the distinct mediolateral 746 683 The nucleus of the stria medullaris appears as a ventrolateral diffusion orientations in the deep layers of the superior colliculus, 747

684 extension of the stria medullaris at the level where the stria medullaris aided also by slightly darker T2* signal and the characteristic reticular 748 685 emerges below the dorsal and anterior surface of the thalamus. The appearance of tissue in the reticular formation. The ventromedial 749 686 nucleus is readily distinguished from the stria medullaris (which has boundary of the superior colliculus was difﬁcult to see in DTI, but 750 687 darker T* contrast) and from the basal forebrain region (which has was approximated by following a wedge-shaped area with brighter 751

688 brighter T2* contrast). T2* signals presumably reﬂecting the precuneiform area. 752

753 The dorsal cap of the inferior colliculus was defined by the brain in the inferior olive further aided delineation against the anteroposterior 812 754 surface and its posterior boundary was given by the precerebellar diffusion orientations in the pyramidal tracts. 813 755 fissure. The gray matter of the inferior colliculus was identified as a 756 large ovoid shaped area with brighter T2* signal than the surrounding Trigeminal nuclei 814 757 precuneiform area and reticular formation. The ventral boundary of The spinal trigeminal nuclei were delineated based on their fairly 815 758 the inferior colliculus was clearly demarcated by the high FA and dorso- bright signal and striated appearance caused by axially oriented fibers 816 759 ventral diffusion orientations of the lateral lemniscus, and more subtle with dark T2* signal. The trigeminal tract (with distinct dark T2* signals) 817 760 differences in T2* signal and DTI orientations in the precuneiform area. delimits the spinal trigeminal nuclei laterally and to a certain extent also 818 761 The medial boundary of the inferior colliculus is given by the commis- dorsally and ventrally. The medial boundary of the spinal trigeminal 819 762 sure of the inferior colliculus, the periaqueductal gray matter and the nuclei was identified on basis of the slightly brighter T2* signals in this 820 763 4th ventricle. region. The principal trigeminal nucleus was not delineated here, and 821 the anterior boundary of the spinal trigeminal nuclei was arbitrarily 822 764 Substantia nigra set at the level where the facial nerve exits the brain stem, while the 823 765 The substantia nigra was delineated as a single structure without posterior boundary was set at the coronal level of the pyramidal 824 766 subdivisions. The lateral and ventral boundaries of the structure are decussation. 825 767 readily given by the cerebral peduncle (with dark T2* signal and high 768 – fi FA; Figs. 4G I), while the dorsal and medial boundaries are more dif - Gray matter in the floor of the 4th ventricle and surrounding the 826 769 cult to see. We interpreted a thin zone with slightly darker T2*contrast central canal 827 770 fl to re ect the somewhat higher cell density of the compact part of the The gray matter found in the continuation of the periaqueductal 828 771 substantia nigra and included this in the dorsomedial boundary of the gray, adjacent to the floor of the 4th ventricle and surrounding the cen- 829 772 substantia nigra. This boundary partly overlaps with the mediolaterally tral canal, was delineated as a continuous structure based on bright T2* 830 773 oriented diffusion orientations located ventral to the medial lemniscus, contrast. 831 774 presumably reflecting fibers emerging from the dorsal part of the 775 substantia nigra. Brain stem 832 In the present atlas, the region brain stem includes all otherwise 833 776 Interpeduncular nucleus unlabeled areas of the midbrain, the pons, and the medulla oblongata. 834 777 The interpeduncular nucleus was readily distinguished from the Accordingly, this region incorporates the reticular formation and several 835 778 brain stem based on its bright T * signal and homogenous and compact 2 nuclei. The anterior boundary of the region is given by the thalamus and 836 779 appearance, as well as its position ventral to the brain stem and rostral hypothalamic region. It is divided medially by the periaqueductal gray 837 780 to the pontine nuclei. and limited dorsally by the tectum and the periaqueductal gray. The 838 ventral borders are given by the descending pathways (cerebral pedun- 839 781 Periaqueductal gray cle and pyramidal tract), substantia nigra, interpeduncular nucleus, 840 782 The periaqueductal gray was delineated on the basis of T2*contrast. pontine nuclei (with bright T2* signal), transverse fibers of the pons 841 783 The anterior boundary was identified on basis of the darker T *signals 2 (high FA values) trapezoid body (dark T2* signal). Further, at the level 842 784Q4 observed in the thalamus (see the Thalamus section, above). In coronal of the pons, the region is framed laterally by the middle cerebellar pe- 843 785 slices, the periaqueductal gray was clearly visible as a region of bright duncle (relatively homogenous dark T2* signal, dorsoventral diffusion 844 786 T2* contrast surrounding the aqueduct (Figs. 4D,E). The posterior end orientation) and the spinal trigeminal tract (very dark T2* signal, distinct 845 787 of the periaqueductal gray and transition to the different tegmental anteroposterior DTI signal). In the medulla oblongata, the dorsal bound- 846 788 nuclei surrounding the 4th ventricle (here included in the brain stem ary is defined by the 4th ventricle and the gray matter adjacent to its 847 789 region) was set at the most anterior level of the cerebellum (where floor (bright T2 and T2* signal), as well as by the cerebellum. The lateral 848 790 the 2nd lobule protrudes into the 4th ventricle). boundary at this level is defined by the inferior cerebellar peduncle and 849 the spinal trigeminal tract, both distinguished based on DTI. Ventral bor- 850 791 Pontine nuclei ders are given by the trapezoid body (mediolateral fiber orientation) 851 792 The pontine nuclei were delineated based on their bright T2*signal and the medial lemniscus (anteroposterior fiber orientation), and fur- 852 793 and their unique position ventral to the brain stem. The present delinea- ther on by the inferior olive (bright T2* signal and distinct shape and 853 794 tion includes the pontine gray matter located ventral to the descending orientation). Posterior to the facial nerve, the region is delimited by 854 795 longitudinal fasciculus of the pons and partly surrounding it. The trans- the spinal trigeminal nuclei appearing laterally (brighter T2*signaland 855 796 verse fibers of the pons and the middle cerebellar peduncles were delin- dotted appearance). Approaching the spinal cord, the brain stem again 856 797 eated separately. has a direct contact with the descending pathway/pyramidal tracts. 857 The posterior boundary of the region is given by the spinal cord. 858 798 Cerebellum 799 The high-resolution T2* images and DT images provided much struc- Ventricular system 859 800 tural detail in the cerebellum (Figs. 4G–I), but we here only subdivided 801 UNCORRECTED PROOF the cerebellum into the molecular cell layer (with very bright T2*con- The ventricular system (comprising the lateral ventricles, 3rd ventri- 860 802 trast) and the deeper cerebellum, which included the Purkinje cell cle, aqueduct, and 4th ventricle) was delineated as a single structure 861 803 layer, granule cell layer, white matter, and deep cerebellar nuclei. The based on the nearly saturated bright signal in T2-weighted and T2*- 862 804 cerebellum was distinguished from the rest of the brain by extrapolat- weighted images, reflecting the presence of the contrast agent used in 863 805 ing the surface of the brain stem through the superior, middle, and infe- this preparation. The same signal was used to delineate the medullary 864 806 rior cerebellar peduncles. At some levels we used an imaginary line and cervical central canal as a separate structure, starting at the obex 865 807 connecting the lateral corner of the 4th ventricle and the lateral recess and extending towards posterior in the whole length of the spinal 866 808 of the 4th ventricle as a guideline. cord delineation. Since the ventricular system is continuous with fur- 867 ther spaces filled with cerebrospinal fluid surrounding the brain, arbi- 868 809 Inferior olive trary boundaries were placed 1) at the anterior level where the pineal 869

810 The inferior olivary complex was delineated based on its bright T2* gland ﬁrst appears in the coronal view, 2) at the posterior level where 870 811 signal and distinct shape. The distinct mediolateral diffusion orientations the pineal gland no longer touches the cerebral cortex on either side 871

872 in the coronal view, and 3) at the posterior level where the horns of the on brain anatomy (see the Results section). Delineation of anatomical 934 873 thalamus (geniculate nuclei) disappear from the coronal view. boundaries in the present atlas therefore required substantial manual 935 segmentation efforts. More than 40 million voxels containing structur- 936 874 Spinal cord al information about the brain was assigned anatomical label using 937 2D and 3D segmentation (with brush diameters ranging from 1 to 938 875 The spinal cord was deﬁned as starting at the last coronal level 88 voxels) by visual interpretation of image features in multiple semi- 939

876 showing the occipital bone. The spinal cord was delineated approxi- transparent layers (T2, T2*, FA, DTI maps) in three orientation planes 940 877 mately 8.62 mm posterior to the skull until leaving the ﬁeld of view. (coronal, sagittal, and horizontal) while taking into consideration the 941

878 The outer boundaries of the spinal cord were identified using bright T2 available atlases and literature. Future datasets registered to our tem- 942 879 and T2* signal originating from the cerebrospinal fluid in the subarach- plate can, however, be subject to “atlas-based” segmentation enabling 943 880 noid space. The central canal and the surrounding gray matter, as well the use of automated routines (see, e.g., Cabezas et al., 2011), as well 944 881 as fibers extending from the descending pathways, were delineated as the generation of a population averaged template (Veraart et al., 945 882 separately. 2011). 946 Atlases are important tools for comparing and analyzing data, and a 947 883 Discussion cornerstone for reaching out to diverse resources containing different 948 types of information about the brain lies in the interoperability of the 949 884 The key results of the present study are fourfold. First, we have pro- spatial reference systems used. We have implemented Waxholm 950 885 duced a microscopic resolution isotropic MRI and DTI template for the Space in our rat brain template, and coupled the template to the stereo- 951 886 Sprague Dawley rat brain. Second, on the basis of this template we taxic skull based coordinate system (Paxinos and Watson, 1982), there- 952 887 have built an atlas of major anatomical structures in the brain, and we by facilitating translation of Waxholm Space to a variety of existing 953 888 provide detailed delineation criteria for all structures in this atlas. atlases operating in stereotaxic space. Interoperability and translation 954 889 Third, we have applied a spatial reference system based on internal between atlases are in the mouse brain managed by a digital atlasing 955 890 brain landmarks according to the Waxholm Space standard, previously infrastructure developed by the International Neuroinformatics 956 891 developed for the mouse brain. Fourth, we have connected this spatial Coordinating Facility (INCF, Hawrylycz et al., 2009, 2011). Principles 957 892 reference system to the widely used stereotaxic coordinate system by and components made available through this infrastructure are equally 958 893 identifying cranial sutures and related stereotaxic landmarks in the suitable and relevant for the rat brain. 959 894 template using contrast given by the active staining technique applied A brain atlas is never complete or finalized. It can always be further 960 895 to the tissue. With the release of the present atlasing template and improved and made more precise. First, the work presented here 961 896 parcellations, we provide a new tool for spatial orientation in analysis contains a relatively robust parcellation of 76 structures, but may well, 962 897 of neuroanatomical location and planning and guidance of experimental at several locations, be subject to different interpretations of boundaries. 963 898 procedures in the rat brain. Second, current ongoing more detailed 3D parcellation of the hippo- 964 899 The atlas is based on contrast enhanced ex vivo MRI acquired using a campal region of the same atlas template demonstrates the potential 965 900 protocol that minimizes morphological distortions by gentle perfusion for further refinement and development of the atlas as a region by re- 966 901 and by imaging the brain in situ within the cranium (Badea et al., gion community effort (Papp et al., 2013). Third, the present atlas tem- 967 902Q5 2007; for comparison with in vivo imaging, see Benveniste et al., plate represents a necessary starting point for creation of population 968 903 2007). It is thus reasonable to assume that tissue fixation has not intro- based probabilistic atlases based on ex vivo or in vivo MRI data from 969 904 duced any major morphological differences in our ex vivo template as additional animals. Other future challenges include further improving 970 905 compared to the in vivo brain, at least not at a level exceeding distor- the understanding of how MRI contrast correlates to histological mea- 971 906 tions that may occur in histological material. Further, the high spatial sures and establishing similar atlas resources for developmental stages. 972 907 resolution and structural contrast obtained in our ex vivo images greatly We provide open access to the presented high resolution template 973 908 facilitate the identification of anatomical landmarks and delineation of and the accompanying atlas. The significance of open access is partly 974 909 smaller anatomical regions that would not be visible in in vivo MRI in the validation, further improvement and expansion of the atlas 975 910 with larger voxel size. Partial volume effects by inclusion of surrounding and partly in providing an atlas that can be freely used with various 976 911 tissues are also reduced by the small voxel size. It should be noted that informatics tools, facilitating for example data analysis with anchoring 977 912 the properties of water diffusion are altered in fixed tissues, such that of experimental data from histology or electrophysiology to atlas space. 978 913 the diffusion parameters in the present data are altered from the 914 in vivo situation, but with relevant diffusion orientations (D'Arceuil Acknowledgments 979 915 and de Crespigny, 2007; Sun et al., 2003, 2005). Earlier rat brain atlasing 916 studies have transferred delineations from existing histological atlases We thank the INCF committee on digital brain atlasing for valuable 980 917 to MRI templates at various levels of granularity. Such atlases have discussions, Christopher Coello and Gergely Csúcs for expert technical 981 918Q6 provided useful results in context of functional (Nie et al., 2012) and assistance, and Sveinung Lillehaug for assistance with anatomical delin- 982 919 pharmacological (Schwarz et al., 2006) neuroimaging, and analyses of eations. This work was supported by grants from the Research Council 983 920 tissue contrast (Johnson et al., 2012). Veraart et al. (2011) used a differ- of Norway and the EC Human Brain Project to J.G.B. MRI imaging was 984 921 ent atlasing approach by delineating anatomical regions directly based UNCORRECTEDperformed at the DukePROOF Center for In Vivo Microscopy, a NIH/NCRR Na- 985 922 on anatomical MRI and DTI in selected regions of the brain. Our work tional Biomedical Technology Research Center (P41 RR005959, to 986 923 expands this concept to the whole brain level. G.A.J.) and Small Animal Imaging Resource Program (U24 CA092656, 987 924 Semi-automatic segmentation of MRI data with the aim of identify- to G.A.J.). 988Q7 925 ing anatomical regions is based on image intensity and contrast, and 926 manual detection of signal changes at locations corresponding to 927 known anatomical boundaries. Changes in intensity and contrast are References 989 928 known to reflect changes in cell and fiber densities (see, e.g. Alexander Aggarwal, M., Zhang, J., Miller, M.I., Sidman, R.L., Mori, S., 2009. Magnetic resonance imag- 990 929 et al., 2007; Mallisch et al., 1991; Norris et al., 2013). But since MRI ing and micro-computed tomography combined atlas of developing and adult mouse 991 930 does not provide the same high level of specificity as histology, transi- brains for stereotaxic surgery. Neuroscience 162, 1339–1350. 992 993 931 tions in signal contrast and intensity were only recorded as boundaries Aggarwal, M., Zhang, J., Mori, S., 2011. 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Please cite this article as: Papp, E.A., et al., Waxholm Space atlas of the Sprague Dawley rat brain, NeuroImage (2014), http://dx.doi.org/10.1016/ j.neuroimage.2014.04.001