NeuroImage 68 (2013) 105–111

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Detection of postmortem human cerebellar cortex and white matter pathways usinghigh angular resolution diffusion tractography: A feasibility study

Emi Takahashi a,b,c,⁎, Jae W. Song a,b, Rebecca D. Folkerth d,e, P. Ellen Grant a,b,c, f, Jeremy D. Schmahmann g

a Division of Newborn Medicine, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston MA, USAb Fetal-Neonatal Neuroimaging and Developmental Science Center, Boston MA, USAc Athinoula A. Martinos Center for Biomedical Imaging, Charlestown, MA, USAd Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston MA, USAe Department of Pathology, Boston Children's Hospital, Harvard Medical School, Boston MA, USAf Department of Radiology, Boston Children's Hospital, Harvard Medical School, Boston MA, USAg Ataxia Unit, Cognitive and Behavioral Neurology Unit, Laboratory for Neuroanatomy and Cerebellar Neurobiology, Department of Neurology, Massachusetts General Hospital, HarvardMedical School, Boston, MA, USA

⁎ Corresponding author at: Division of Newborn MedBoston Children's Hospital, Harvard Medical School, US

E-mail address: emi@nmr.mgh.harvard.edu (E. Taka

1053-8119/$ – see front matter © 2012 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.neuroimage.2012.11.042

a b s t r a c t

a r t i c l e i n f o

Article history:Accepted 21 November 2012Available online 11 December 2012

Keywords:BrainCerebellumHumanDiffusion imagingTractography

Imaging three-dimensional cerebellar connectivity using diffusion tractography is challenging because of theubiquitous features of crossing axonal pathways within a folium as well as intersecting pathways from neigh-boring folia. We applied high-angular resolution diffusion imaging (HARDI) tractography to intact postmor-tem adult brainstem and cerebellum to examine the 3-dimensional white matter and local gray matterpathways. The middle cerebellar peduncles conveyed fibers from the rostral pons to the lateral and caudalaspects of the cerebellar hemisphere, and from the caudal pons to medial and rostral parts of the cerebellarhemisphere. In the cerebellar cortex, tractography detected tangential coherence superficially in the cerebel-lar cortex and revealed fibers coursing parallel to the long axis of the folia. These fibers were consistent withthe location and direction of parallel fibers in the molecular layer. Crossing with these parallel fibers weretangential fibers running perpendicular to the long axis of the folia, consistent with axons of the corticalinterneurons — stellate cells and basket cells. These tangential fibers within the cerebellar cortex were dis-tinct from the fibers linking the cerebellar cortex with the deep cerebellar nuclei and the brainstem. Our re-sults show the potential for HARDI tractography to resolve axonal pathways from different neuronalelements within the cerebellar cortex, and improve our understanding of adult cerebellar neural circuitryand connectivity in both white and gray matter.

© 2012 Elsevier Inc. All rights reserved.

Introduction

The cerebellum is critical for sensorimotor function, as well as forintellectual processing and emotional regulation (Schmahmann, 1997,2010). Anatomical studies of the cerebellum and its connections withthe cerebral hemispheres in the non-human primate have helped de-fine the neurobiological underpinnings of these relationships (Brodaland Bjaalie, 1997; Schmahmann and Pandya, 1997; Strick et al., 2009).The development of diffusion tractography enables the study of theseconnections non-invasively in animal models and in the human brain.Diffusion tensor imaging (DTI) studies of the developing cerebellum(Huang et al., 2009; Saksena et al., 2008) have shown the location andtrajectories of the major cerebellar white matter pathways, and thesenew imaging approaches hold the promise of a deeper understandingof the anatomical basis of cerebellar functions in the human brain.

icine, Department of Medicine,A.hashi).

rights reserved.

Identification of the complex 3-dimensional patterns of intracerebellarand extracerebellar connections using diffusion tractography is limited,however, because of the multiple crossing axonal pathways in the cer-ebellar white matter and cortex, and because detection of tractographypaths within a folium is often contaminated by paths from the closelyapposed and narrow neighboring folia.

High-angular resolution diffusion imaging (HARDI) improves thecharacterization of complex tissue coherence compared to DTI by virtueof its ability to define fiber orientation distribution functions (Leergaardet al., 2010; Tuch et al., 2003). This approachmakes it possible to resolvedifferent diffusion directions within the same voxel resulting fromcrossing axonal bundles. We have previously used HARDI tractographysuccessfully to resolve white matter pathways in the cerebellum in vivo(Granziera et al., 2009) and in the cerebrum ex vivo (Takahashi et al.,2011, 2012). High-resolution tractography using HARDI has not yetbeen applied to the study of postmortem human cerebellum. In thisstudy our objectivewas to apply HARDI tractography to intact postmor-tem adult human cerebellum to examine the 3-dimensional fiber struc-tures in cerebellar white matter and local gray matter.

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The advantages of ex vivo imaging include the lack of motion thatenables imaging at very high resolution for the long periods of timenecessary to obtain adequate signal to noise for HARDI, the absenceof blood flow as a confounding factor, and the lack of susceptibility ar-tifacts at air or bone and brain interfaces. These advantages of ex vivoimaging are critical when trying to image fibers in the cerebellar cor-tex because the cortex is thin, and because fibers in the cerebellarcortex are less coherent than those in the white matter. HARDI iswell-suited for identifying pathways in the cerebellar gray matter be-cause different types of cells are regularly aligned in the cerebellarcortex and the topographical relationships between the cells andfolia are common across regions. In addition to the advantages ofimaging postmortem tissues and the naturally coherent and regularcellular organization of the cerebellum, an optimal MR coil can substan-tially improve signal to noise and the ability of diffusion tractography toidentify gray matter structure. MR signal strength is proportional to thedistance from the MR coil. The location of the cerebellum within thewhole brain therefore presents a challenge to obtaining optimal signalto noise and spatial resolution. In this postmortem study we dissectedthe cerebellum and brainstem off from the cerebrum, and used an MRcoil closely apposed to the surface of the cerebellum, thus enablingthe highest possible signal to noise ratio and spatial resolution.

Experimental procedures

Specimens

We performed MR scans on three specimens of the adult humancerebellum. The cerebellums were acquired from the Brigham andWomen's Hospital, Department of Pathology, under protocols approvedby the hospital's institutional review board for human research. A neu-ropathologist studied each brain at the time of post-mortem examina-tion, and only those not needed for immediate neuropathologicaldiagnosis were fixed in 4% paraformaldehyde and submitted for coded(de-identified) specimen scanning. Mean fixation period was approxi-mately 2–3 months. Any cases with known or suspected malformations,disruptions, or other lesions were excluded from this study. Brains wereremoved from the cranium and fixed in a 4% paraformaldehyde solutioncontaining 1 mM gadolinium (Gd-DTPA) MRI contrast agent for at least1 week to reduce the T1 relaxation time while ensuring that sufficientT2-weighted signal remains. Before image acquisition, the cerebellumand pons were separated from the cerebral hemispheres by transversesection through the midbrain at the level of the colliculi. During imageacquisition, the cerebellumwith pons attachedwas placed in Fomblin so-lution (Ausimont, Thorofare, NJ) and imaged on a 4.7 T Bruker BiospecMR system, using an MR coil (10 cm diameter) in order to maximizethe signal to noise ratio.

Scanning parameters

The pulse sequence used for HARDI acquisition was a 3D diffusion-weighted spin-echo echo-planar imaging (EPI) sequence, TR/TE1000/40 ms, with an imaging matrix of 128×128×112 pixels. Sixtydiffusion-weighted measurements and one non diffusion-weightedmeasurement were acquired at b=4000 s/mm2 with δ=12.0 ms,Δ=24.2 ms. Spatial resolution was 525×525×600 μm. The total ac-quisition time was approximately 1 h and 50 min for each imagingsession.

Diffusion data analyses — tractography

Weused a streamline algorithm for diffusion tractography (Mori et al.,1999) described in previous publications (Takahashi et al., 2011, 2012).The term “streamline” refers to the fact that we connect tractographypathways using local maximum or maxima. This is true for both DTIand HARDI. The streamline technique is limited in its ability to resolve

crossing pathwayswhenusedwith the traditional DTI technique, becauseone simply connects the direction of the principal eigenvector on a tensorto produce the DTI tractography pathways. This is a recognized limitationof DTI, as discussed in the DTI paper of Mori et al. (1999). For this reason,in the current study, we used HARDI, which detects multiple local maxi-ma on an ODF (orientation distribution function). We used all the localmaxima to produce HARDI tractography pathways, thus enabling us toidentify crossing pathways within a voxel.

Trajectories were propagated by consistently pursuing the orienta-tion vector of least curvature. We terminated tracking when the anglebetween two consecutive orientation vectors was greater than thegiven threshold (45°). In many tractography studies, FA values areused to terminate fibers in the gray matter. In adults, the FA values ofthe gray matter are lower than in the white matter. However, becauseone of the objectives was to detect fibers in low FA areas (cerebellarcortex), we used mask (boundary) images of the brains created byMRIcro (www.sph.sc.edu/comd/rorden/mricro.html) in order to deter-mine coherence in the brain itself and not in the surrounding immersionfluid. Of note, themask image is a 3-dimensional (3-D) binary image cre-ated from a mean diffusion image of each brain assessment with end-points constrained to the brain tissue itself. This process is routinelyused to remove skull images or susceptibility-related artifacts aroundthe brain surface. Thismethodhas beenused previously and is an accept-able alternate method (e.g. Schmahmann et al., 2007; Takahashi et al.,2012; Vishwas et al., 2010; Wedeen et al., 2008).

Diffusion Toolkit and TrackVis (http://trackvis.org) were used toreconstruct and visualize tractography pathways. The color-coding oftractography pathways in Fig. 1 (left panel), Figs. 3, 4A, and 5 are basedon a standard RGB code, applied to the vector between the end-pointsof each fiber. Right panels of Figs. 1 and 4were color-coded for FA values.In Fig. 2, red identifies the cortico-spinal tract and blue the superior,green the middle, and yellow the inferior cerebellar peduncle pathways.In Fig. 6, orange identifies a part of the ponto-cerebellar pathways, blueidentifies pathways running parallel to the long axis of folia, and greenidentifies pathways running perpendicular to the blue pathways.

Region of interests (ROIs) and filters for identifying tractography pathways

To improve visualization in the figures, we restricted the number oftractography pathways displayed in the following ways. For Figs. 1, 4,and 5, we used sagittal (Figs. 1 and 4) or axial (Fig. 5) slice filters. Thethickness of the slice filters was 3 pixels and 50% of the total pathwaysrunning through the slices were displayed using standard options inTrackVis. For Fig. 2, we used at least two ROIs to identify each fiber path-way (pons and spinal cord for the corticospinal pathways, posteriorpons and deep cerebellar white matter for the superior cerebellar pe-duncle, anterior pons and cerebellar hemisphere for themiddle cerebel-lar peduncle, and posterior pons and cerebellar hemisphere for theinferior cerebellar peduncle). For Fig. 3, we used only one ROI in thepons. The position of the ROI is shown in the lower left corner of the fig-ure. For Fig. 6, we used one ROI in the pons to identify yellow or whitepathways and used another ROI in the cerebellar cortex to identifylocal cortical pathways. The positions of the ROIs are shown in thelower left corner of Fig. 6A.

Results

Cerebellar white matter

Cerebellar white matter showed high FA values both in deep, thickbundles in the medullary core and in the branched thinner laminaeextending out to the folia that constitute the cerebellar cortex(Fig. 1, asterisk). More than 98% of the total detected white matterpathways showed FA values ranging from 0.3 to 0.53. The superior(Fig. 2, dark blue), middle (Fig. 2, green), and inferior (Fig. 2, yellow)cerebellar peduncles were clearly identified along with the corticospinal

Fig. 1. Directional and FAmaps of pathways running through a sagittal slice. Cerebellar white matter showed very high FA values both in deep, thick bundles of pathways and also inbranched thinner pathways in the cerebellar cortex (asterisk). A: anterior, P: posterior, D: dorsal, V: ventral, L: left, R: right.

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tract (Fig. 2, red). The middle cerebellar peduncle conveyed fibers fromthe rostral pons to the lateral parts of the cerebellar posterior lobe(Fig. 3, predominantly light blue) and from the caudal pons to medialand intermediate regions of the superior aspect of the cerebellar hemi-sphere (Fig. 3, predominantly green).

Cerebellar cortex

On the FA images, the cerebellar folia had3 apparent layers. In the sag-ittal plane perpendicular to the folia, the most superficial “layer” showedhigh FA values (>0.20) (Fig. 4a), the apparent second layer showed verylow FA values (0–0.05) with less pathways (Fig. 4b), and the apparentthird layer showed medium degrees of FA values (0.05–0.10) (Fig. 4c).The first and second “layers” were 3–4 mm each, and the third “layer”was about 2 mm. The thicknesses and the extent of the apparent layerssuggested that these layers result from volume averaging of pathwaysfrom adjacent folia because the spatial resolution of the images is greaterthan the thickness of the cortex of the folia (see further discussion in thesection on Limitations and advantages of the current study).

In the axial planewe successfully detected pathways running parallelto the folia in the gray matter (Fig. 5a, panels A–C; green pathways) thatwere distinct from pathways running in the white matter (Fig. 5b, panelC; red pathways). The anatomic images upon which the tractographypathways are superimposed are the mean diffusion images in whichgray matter is seen as white, and white matter is gray (Fig. 5, panel B).Consequently, the pathways coursing parallel to the folia are evident inthe lighter appearing gray matter, whereas the pathways lying in thewhite matter are visible in the gray regions in these mean diffusion im-ages (Fig. 5, panel B).

We next examined the topographic organization of the differenttypes of projection pathways. A low magnification view of the right

Fig. 2. Identification of white matter pathways. Left panel shows a lateral view, and the rigcerebellar peduncles, and the corticospinal tract (red). Small insets show each pathway sep

hemisphere of an adult cerebellum in the axial plane is shown inFig. 6A. The yellow box indicates a region of Lobule VIIA, Crus I, whichis magnified in Fig. 6B. Orange pathways were observed perpendicularto both blue and green pathways. These orange pathways extendedfrom the pons to the cortical layer of the cerebellum (Fig. 6A and B).Blue tractography pathways were observed running in a direction paral-lel to the folia, and green fiber pathwayswere observed running perpen-dicular to, but in the same plane as the blue pathways (Fig. 6C and D).

Fig. 6E illustrates the cytology and circuitry of the cerebellar cortex.Ascending granule cell axons, parallel fibers, basket cell axons, and stel-late cell axons are situated in the molecular layer. Parallel fibers courseparallel to the long axis of the folia, whereas axons of the cerebellar cor-tical inhibitory interneurons (stellate cells in the mid and upper reachesof themolecular layer, and basket cells in the lower part of themolecularlayer) are oriented perpendicular to the long axis of the folia. Therefore,the blue tractography pathways in Fig. 6A–D likely correspond to groupsof parallel fibers, and the perpendicularly oriented green tractographypathways in Fig. 6A–D likely represent the axons of stellate and basketcells. The orange tracks in Fig. 6A–B represent afferents to the cerebellarcortex including the mossy and climbing fibers, as well as nucleocorticalrecurrent collaterals; and efferents from the cerebellar cortex conveyedby the Purkinje cell axons.

Discussion

Our results show that HARDI tractography is able to visualize knownstructures within the white matter and cortex of the adult human cere-bellum post-mortem. In particular, our results demonstrate coherentfiber track structure likely reflecting the axonal pathways that charac-terize different cell types within the cerebellar cortex.

ht panel shows a medial view of the superior (blue), middle (green), inferior (yellow)arately. A: anterior, P: posterior, D: dorsal, V: ventral, L: left, R: right.

Fig. 3. Topograpy of the middle cerebellar peduncle projections. The middle cerebellar peduncle conveyed fibers from the rostral pons to the lateral aspects of the cerebellar pos-terior lobe (mostly light blue pathways) and from the caudal pons to medial parts of the superior aspect of the cerebellar hemisphere (mostly green pathways). Region of interests(ROIs) for identifying pathways are shown in the lower left corner. A: anterior, P: posterior, D: dorsal, V: ventral, L: left, R: right.

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Topical connections conveyed in the middle cerebellar peduncle

To our knowledge, this is the first imaging demonstration of differen-tial pontocerebellar projections in the middle cerebellar peduncle. Fibertracks from the rostral pons are directed to the lateral and caudal aspectsof the cerebellar hemisphere (Fig. 3, mostly light blue), whereas thosefrom the caudal pons are identified in medial and rostral parts of thecerebellar hemisphere (Fig. 3, mostly green). This rostral pons to poste-rior cerebellum, and caudal pons to anterior cerebellum connectionwasevident in early myelin stain studies (von Bechterew, 1885) and ana-tomical investigations (Spitzer and Karplus, 1907; Sunderland, 1940;see Schmahmann et al., 2004), and is consistent with anatomical, func-tional imaging, and clinical evidence pointing to a gradient of caudalpons – anterior lobe of cerebellum sensorimotor representation as op-posed to a rostral pons – posterior lobe of cerebellum cognitive trend(Schmahmann and Pandya, 1997; Schmahmann and Sherman, 1998;Schmahmann et al., 2009; Stoodley and Schmahmann, 2010; Buckneret al., 2011).

There are several possible reasons why we were able to detect differ-ential arrangement of pontocerebellar fibers in the middle cerebellarpeduncle. Ex vivo imaging provides the benefit of very high spatial resolu-tion and high signal-to-noise-ratio because artifacts due to movementand air are minimized. Further, the method of specimen preparation,using a size-optimized sample container and MR coil, and HARDItractography reconstruction further improved the quality of the resultingtractography pathways (see Takahashi et al., 2010). Given that pathways

Fig. 4. Directionally color coded tractography and FA maps of the cerebellar folia with tractshowed high FA values (a), the second layer showed very low FA values with fewer tracts idfolia and subfolia showing that tractography pathways are continuous across subfolia likeaveraging across this boundary.

in the same bundle (in this case the middle cerebellar peduncle) tend torun almost in parallel in a small region (in other words, in adjacentvoxels), high-angular resolution is needed to dissociate such pathways.A clear dissociation of projection patternswithin a bundlemaybe difficultto detect in vivo, with DTI, or using un-optimized experimental settings.

Potential for HARDI tractography to resolve axonal pathways from differenttypes of cells within the cerebellar cortex

HARDI tractography detected tangential coherence superficially inthe cerebellar cortex, displaying fibers coursing parallel to the longaxis of the folia. The location and direction of these fibers are consis-tent with the well-defined features of parallel fibers in the molecularlayer. Crossing these putative parallel fibers were tangential fibersrunning perpendicular to the long axis of the folia, consistent withthe axons of stellate cells in the mid-and upper regions of the molec-ular layer, and of the basket cell axons in the lower part of the molec-ular layer. These tangential fibers within the cerebellar cortex weredistinct from the fibers linking the cerebellar cortex with the deepcerebellar nuclei and brainstem.

The ascending granule cell axon divides to form the parallel fiberswhich course along the long axis of the cerebellar folium. The branchesof each parallel fiber branch travel for a distance of approximately3 mm in the rat and up to 8 to 10 mm in the human. In the rat, the basketcell axon is approximately 350–400 μm and synapses with the somataand proximal axons of 9 to 10 Purkinje cells (Palay and Chan-Palay,

s restricted to those remaining within the sagittal slice. A, B. The most superficial layerentified (b), and the third layer showed medium FA values (c). C. Schematic example ofly due to the spatial resolution of the images being 0.525 to 0.600 mm and therefore

Fig. 5. Pathways in the cerebellar cortex running through an axial slice. A, B. Views from the top with low intensity (A) and high intensity (B) mean diffusion images. C. A view fromthe bottom with high intensity mean diffusion image. Pathways running parallel to folia were detected in the gray matter (a in panel A, B, and C), and they were distinct from path-ways running in the white matter (b in panel C). A: anterior, P: posterior, D: dorsal, V: ventral, L: left, R: right.

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1974). In the human data in our study, the tractography pathways sug-gestive of parallel fibers (blue pathways in Fig. 6) ranged from 3 to10 mm, and the axons of stellate and basket cells (green pathways inFig. 6) ranged from 1 to 10 mm. Tractography pathways representmeanwater diffusivity in a direction within a voxel. It is possible there-fore, that each tractography path represents multiple, similarly alignedparallel fibers or axons of interneurons within a voxel, linking differentaxon trajectories end-to-end and resulting in a spuriously long course.

Limitations and advantages of the current study

Angle thresholds to terminate tractography fibers are arbitrarilyset in many tractography studies (typically ranging from 35°–60°)to minimize false positive pathways in some brain areas.

This approach is potentially misleading because an arbitrary settingthat is optimal in one region may be suboptimal in another, resulting infalse fiber tracks. Given the complexity of fiber circuitry and regionaldifferences in the brain, future studies should investigate methods ofoptimizing angle thresholds and other regionally variable tractographyparameters (see also discussion of Takahashi et al., 2010). We were notable to identify fiber tracks in subfolia as clearly as those in the mainfolia, a limitation that may be related to the angle threshold setting, aparameter selected by investigators for tractography.We used a thresh-old of 45° for tractographywhichwould fail to detect those fibers in themain stem of a foliumwhich turn sharply (almost 90°)when entering asubfolium. We do not currently use an angle threshold of 90° so as tolimit the likelihood of reconstructing spurious tracks, but in order tobetter image pathways in subfolia it may be necessary in future studiesto set different angle thresholds in different brain regions.

We successfully identified tractography pathways likely corre-sponding to structural coherence arising from groups of parallel fibersand axons of interneurons in the axial plane (Fig. 5), while in the sag-ittal plane we observed spurious laminar organization in the cerebel-lar cortex (Fig. 4A–B). Pathways corresponding to parallel fibers andaxons of interneurons indeed existed in sagittal views, but were

difficult to visualize in that plane using only a sagittal slice filter(Fig. 5). It was necessary to use a spherical ROI in the cerebellar cortexin addition to the sagittal slice filter to identify the pathways in the sag-ittal plane (Fig. 6). Based on the thickness and broad expansion of thelayers parallel to the cerebellar surface, the apparent layers in Fig. 4 donot seem to be actual cell layers of the cerebellar cortex, but rather amix-ture of real fiber pathways across adjacent folia (Fig. 4C). As evident inthe folial structures exemplified in Fig. 4C, the first layer could representfibers in the surface of the cerebellum, the second layer could representgaps between subfolia belonging to different folia, and the third layercould represent fibers in deep subfolia adjacent to each other. Giventhat the spatial resolutionwas similar (525–600 μm) in the axial, sagittal,and coronal directions, all directions have similar degrees of difficultywhen performing tractography.

Coherent tractography pathways likely corresponding to parallel fi-bers were clearly visualized in axial views (Fig. 5), but the pathwayswere not well seen in sagittal views (Fig. 4) mainly because they wereshort and running perpendicularly through the sagittal planes. Similarly,tractography pathways likely corresponding to axons of interneuronswere clearly visualized in sagittal views (Fig. 4, layer a), but not in axialviews (Fig. 5). Therefore it was important to analyze pathways by view-ing them in all planes, by rotating them in a three dimensional manner,and often by making another ROI within the cerebellar cortex (Fig. 6).

An advantage of our study is that we have the potential to detectpathways in subfolia. The spatial resolution of our HARDI imaging was500–600 μm, while a single subfolium is about 3–4 mm along the shortaxis (Schmahmann et al., 2000). Given that the thickness of the graymat-ter is less than 1 mmon each side of the short axis (Schmahmann, 2007),and the thickness of the white matter in a subfolium is about 2 mm, thisshould be sufficient to detect with a spatial resolution of 3–4 voxels.These studies are ongoing.

We were not privy to the gender of the individuals from whom thebrains in our studywere obtained. Sexual dimorphism of thalamus, cor-pus callosum and cingulum has been shown in diffusion MRI studies ofthe human cerebrum (e.g. Menzler et al., 2011), and additional studies

Fig. 6. A. A low magnification view of the right hemisphere of an adult cerebellum in theaxial plane. Region of interests (ROIs) for identifying pathways are shown in the lowerleft corner. The yellow box shows a region of Crus I, which is magnified in Fig. 6B. B. Bluetractography pathwayswere observed running in a direction parallel to the folia in a super-ficial plane. In the same plane, green pathways traversed in a perpendicular direction to theblue pathways. Orange pathways were observed running perpendicular to these blue andgreen pathways. This pattern of tractography pathways was observed in many regions ofthe cerebellum. C–D. The relationship of the blue and green fiber pathways is exemplifiedin these two panels. Many of the green fiber pathways were situated within and belowthe blue fiber tracks. This spatial relationship suggests that the blue pathways representparallelfibers, and thegreen tracks represent axons of stellate and basket cells interneurons.E. An illustration of transverse and longitudinal sections of the cerebellum cortex. Note thataxons of basket cells and stellate cells (greenfibers, green arrows) run perpendicular to par-allel fibers (blue fibers, blue arrows for example). The longitudinally oriented pathways inthegranule cell layer comprisemultiple types of afferent and efferentfiber pathways. A: an-terior, P: posterior, D: dorsal, V: ventral, L: left, R: right.

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with the gender informationwill be needed to determinewhether gen-der effects cerebellar white and gray matter pathways as well.

Relevance to future in vivo or validation studies

One of the strengths of our study is the demonstration of the3-dimensional structure of cerebellar white matter pathways. Further,to our knowledge this is the first study to depict the 3-dimensional

structure of the cerebellar cortex with this degree of detail. These find-ings contribute to a more thorough understanding of the organizationof cerebellar circuits and pathways in healthy individuals, and theyhave the potential to shed light on future studies in patients with disor-ders of the cerebellum.

OurMRI diffusionmeasures are volume averages over 500–600 μm3

and at present we cannot identify micro-structural features below thisscale. The observed global view of diffusion coherence in the cerebellarcortex is nevertheless important as we are able to detect crossing path-ways in the cerebellar cortex that have not previously been appreciatedusing MRI. These results will be relevant for future in vivo studies in pa-tients, and they also facilitate future immunohistochemical validationstudies (e.g. Xu et al., in press) of the observed imaging findings in post-mortem cerebella from healthy controls and from patients with cerebel-lar disease. By building on the findings described here, future in vivodiffusion tractography studies identifying damage to the cerebellar cor-tex and white matter have the potential to provide new insights intothe anatomic pathophysiology and temporal progression of the degener-ative ataxias and also to the cerebellar component of neuropsychiatricdisorders.

It is essential to develop a clear picture of the normal patterns andtiming of development of cerebellar pathways and to interpret the roleof white matter pathways in order to more accurately diagnose subtledisorders of cerebellar connectivity. At this point, the ability to applyour methods to the in vivo population is limited by motion and possiblespatial resolution, however, progress is being made in that major path-ways are beginning to be clearly identified (Granziera et al., 2009). Thevisualization of the true complexity of a structure is crucial for under-standing functionally important trajectories, and these are accurately re-vealed onlywhen one visualizes the complex structure in its entirety. Toidentify functionally important connectivity among all of the anatomicalpathways detected, it will be important for future studies to combine invivo MR diffusion tractography with functional MR connectivity.

Acknowledgments

This work was supported by the Eunice Shriver Kennedy NationalInstitute of Child Health and Development (NICHD) (R21HD069001)(ET) and National Institute of Mental Health (R01MH06044) (JDS).This researchwas carried out in part at the Athinoula A.Martinos Centerfor Biomedical Imaging at the Massachusetts General Hospital, usingresources provided by the Center for Functional Neuroimaging Technol-ogies, P41RR14075, a P41 Regional Resource supported by the Biomed-ical Technology Program of the National Center for Research Resources(NCRR), National Institutes of Health. This work also involved the use ofinstrumentation supported by the NCRR Shared Instrumentation GrantProgram (1S10RR023401, 1S10RR019307, and 1S10RR023043) andHigh-End Instrumentation Grant Program (S10RR016811).

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