Mapping of ﬁber orientation in human internal capsule by ... · The internal capsule was parcellated in the areas CI 1 to CI 4 containing ﬁbers of distinct orientation and structure,

Journal of Neuroscience Methods 94 (2000) 165–175

Mapping of fiber orientation in human internal capsule by meansof polarized light and confocal scanning laser microscopy

Hubertus Axer *, Diedrich Graf v. KeyserlingkInstitut fur Anatomie I, Uni6ersitatsklinikum der RWTH Aachen, Pauwelsstraße 30, 52057 Aachen, Germany

Received 8 December 1998; received in revised form 13 August 1999; accepted 1 September 1999

Abstract

The nervous fibers in the human internal capsule were mapped according to their three-dimensional orientation. Four humancadaver brains were cut into comparable and standardized sections parallel to the ACPC-plane, stained with DiI, and analyzedusing a combination of confocal and polarized light microscopy at the same time. This combination provides information aboutthe structure and orientation of the fibers in great detail with confocal microscopy, and information about the localization andorientation of long myelinated fiber tracts with polarization microscopy. The internal capsule was parcellated in the areas CI 1to CI 4 containing fibers of distinct orientation and structure, which enriches the macroscopically definable parcellation in theanterior and posterior limb. Fibers of the anterior thalamic peduncle intermingle with frontopontine tract fibers. Single fibersconnect the caudate and the lentiform nucleus. The pyramidal tract is located in the anterior half of the posterior limbintermingled with fibers of the superior thalamic peduncle. Parietooccipitopontine fibers are located in the posterior part of theposterior limb. The slopes of the different systems of fibers change continuously in the anterior–posterior direction of the internalcapsule. Using the 3D orientation of fibers as a criterion for parcellation, as well as the description of bundles as a collection offibers belonging to particular tracts leads to a more function-related description of the anatomy of the internal capsule. Themethod can be used for interindividual, sex- or age-related comparisons of particular systems of fibers. © 2000 Elsevier ScienceB.V. All rights reserved.

Keywords: Fiber mapping; Internal capsule; Confocal; DiI; Polarization; Corticopontine tract; Pyramidal tract; Thalamic radiation

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1. Introduction

The fiber architecture especially of the white matterin the human brain is presently the focus of interest.Many neurologic diseases affect the cerebral white mat-ter, and modern imaging procedures such as magneticresonance imaging (MRI) provide detailed visual infor-mation about the white matter. Anatomical knowledgeabout nervous fibers was achieved earlier by fiberpreparations (Ebeling and Reulen, 1992), myelogenicstudies (Kretschmann, 1988) and by observations afterbrain injury (Fries et al., 1993).

We were able to demonstrate the orientation andtexture of the nervous fibers to influence the value ofimpedance measurements, and so an online verification

of the needle’s position in a stereotactic procedure canbe performed (Axer et al., 1998). In addition, theorientation of nervous fibers can be visualized by mod-ern MRI procedures (Curnes et al., 1988; Douek et al.,1991; Peled et al., 1998). Nevertheless, a detailedanatomical mapping of fiber orientations in the adulthuman brain is difficult to assess. Our aim was toperform a mapping of the orientation of the fibers inthe internal capsule. Thus we wanted to find out howanatomically defined fiber tracts are oriented in thewhite matter. The method should give informationabout fiber texture, fiber orientation and identificationof specific fiber tracts.

Therefore we used the combination of confocal lasermicroscopy and polarized light microscopy. The confo-cal laser microscope allows information to be collectedfrom well-defined optical sections. This is done by asequential illumination which is focused on one volumeelement of the specimen at a time (Wright et al., 1993).

* Corresponding author. Tel.: +49-241-8089100; fax: +49-241-8888431.

E-mail address: [email protected] (H. Axer)

0165-0270/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 5 -0270 (99 )00132 -6

H. Axer, D.G. Keyserlingk / Journal of Neuroscience Methods 94 (2000) 165–175166

This way stacks of optical sections can be produced,which allow three-dimensional reconstruction of thefiber texture and provide good information about theorientation of the fibers at high resolution.

Polarized light microscopy can selectively visualizeanisotropic structures. In polarization microscopy, opti-cally polarized light passes through a sample of tissueand into a second polarizer (analyzer), which polarizeslight in a perpendicular plane with respect to the firstpolarizer. Birefringence is able to twist some of the lightso that it can pass through the analyzer and be imaged.The birefringence of the nervous tissue has been wellknown for a long time (Schmidt, 1923, 1924; Schmittand Bear, 1936; Kretschmann, 1967; Wolman, 1975).As the presence of anisotropy indicates polarity andorder, polarization microscopy can be used to visualizelong fiber tracts in the brain (Fraher and MacConnaill,1970; Miklossy and Van der Loos, 1991). The orienta-tion of the fibers influences the transmission of plane-polarized light at different velocities at differentazimuths.

The combination of both techniques allowed us toobtain detailed information of the fiber structure andorientation with confocal microscopy and to collectinformation about the localization and orientation oflong myelinated fiber tracts with polarized lightmicroscopy.

The internal capsule in the adult human brain repre-sents a collection of different systems of fibers closelylocated in a small space. It is a structure of high clinicalimportance, since the fibers of the pyramidal tract arelocated here. Nevertheless the exact location of thepyramidal tract in the internal capsule was a matter ofdispute for decades (Maurach and Strian, 1981).

2. Materials and methods

2.1. Macroscopic preparation

Four human cadaver brains without macroscopicallydefinable pathology and without a history of neurologicor psychiatric disease were fixed in 4% aqueous forma-lin solution and macroscopically prepared. Themeninges were removed and the brains cut in themedian-sagittal plane. Important landmarks were iden-tified on the median surface of the hemispheres: theanterior and posterior commissure, the interventricularforamen of Monro and the internal cerebral vein (orthe tela choroidea ventriculi tertii). The next two sec-tions were cut parallel to the line between anterior andposterior commissure (ACPC). The first horizontal sec-tion was made at the level of the internal cerebral vein.The second section was made at the level of the inter-ventricular foramen in the same direction. This way theinternal capsules in all eight hemispheres were sectionedin comparable planes.

2.2. Histologic preparation

The slabs were first placed for 24 h in 10% sucrosesolution and then in 30% sucrose solution until theysank to the bottom of the container. Afterwards thetissue was sectioned on a freezing microtome at 60 mmin the horizontal plane. This way all histologic sectionswere orientated parallel to the ACPC-line. The sectionswere serially collected. Every second section was cover-slipped without staining procedure. The other sectionswere kept in Tris buffer (0.1 M, pH 7.4) containing0.1% Triton™ X-100 (Merck, Darmstadt, Germany)for 24 h. Afterwards these sections were placed onmicroscope slides and 3–4 drops of a DiI-solution (1mg DiI [1,1%-dilinoleyl-3,3,3%,3%-tetramethylindocarbo-cyanine perchlorate, FAST DiI™ oil, Molecular ProbesEurope, Leiden, Netherlands] in 1100 ml dimethylsul-foxide [DMSO]) were put on these slides, and then theslides were kept in a moist chamber at 37°C. DiI ishighly lipophilic and dissolves into lipid membranes.This way the myelin sheaths of the nerve fibers inparticular are labeled. After 7 days, the sections weremounted with the aqueous mounting medium Aqua-tex™ (Merck, Darmstadt, Germany) and coverslipped.

2.3. Con6entional polarized light microscopy

The unstained sections were analyzed by conven-tional polarized light microscopy. We used the StemiSV 11 (Zeiss, Germany) with rotatable crossed polarsfor low magnification. This way we obtained a map ofthe entire sample. A Leica DM IRBE (Leica Microsys-tems, Heidelberg, Germany) microscope equipped witha rotatable stage and fixed crossed polars was used forhigher magnification. The same microscope was usedfor confocal laser microscopy.

2.4. Confocal laser scanning microscopy

The DiI stained sections were analyzed with theLeica TCS NT confocal laser scanning microscope (Le-ica Microsystems, Heidelberg, Germany). Since DiI(maximum absorption: 549 nm, maximum emission:564 nm) has fluorescent features similar to rhodaminewe used the standard rhodamine filter settings of theconfocal microscope.

2.5. Polarization with transmitted laser light

Polarized light microscopic pictures were producedon a second channel of the confocal microscope. There-fore the transmission mode of the microscope was used.In transmission mode the laser light passes through thesample and is detected in a photomultiplier at the otherside of the sample. Since laser light is polarized we hadto insert one polarization filter (analyzer) only into the

H. Axer, D.G. Keyserlingk / Journal of Neuroscience Methods 94 (2000) 165–175 167

Fig. 1. Combination of confocal and polarization microscopy.

after rotation of 45°. The steeper these tracts of fibersare, the less bright the signal is. Thus different fibertracts of different orientation and order produce areasof different brightness. In low magnification, the polar-ization picture provides information about the localiza-tion of different bundles of fibers (Fig. 2).

At higher magnification single bundles of fibers canbe detected. In polarization mode, the area resemblinga bundle of fibers is a summation over the wholethickness of the section (60 mm), as the light must passthrough the entire sample. At the same time confocalimaging allows the identification of single fibers andtheir texture (Figs. 3–6). The image produced with theconfocal microscope is a small optical section in thesample. The angle of the fibers was defined by 3D-re-constructions from confocal z-series (Fig. 7).

3.1. A re6ised parcellation of the internal capsule

Fig. 8 shows the parcellation of the internal capsulebased on the distribution of differently oriented fibers.These areas are different from the borders of themacroscopically defined parcellation of the internalcapsule (anterior limb, genu, posterior limb). The inter-nal capsule was divided into four areas CI 1 to CI 4 fora better orientation.

Three distinct fiber systems can be distinguished inthe anterior limb. Many fibers originate from the thala-mus and run in the direction of the anterior limb. Inour sections these fibers are cut almost horizontally(Fig. 3) and are located in the whole area of the

path of the laser beam after the light has passedthrough the sample. This way it was possible to analyzea single section with both confocal fluorescence andpolarization methods (Fig. 1) at the same time.

3. Results

In polarization microscopy, the detectable signal de-pends on the order and the inclination of the fibers. Inparticular, parallel, horizontally cut fibers give a brightsignal at a special azimuth whereas the signal decreases

Fig. 2. Anterior limb of the internal capsule as revealed by conventional polarization microscopy. Systems of fibers of different orientation appearin characteristic scales of grey under this azimuth. The rectangle in the map in the right upper corner shows the orientation of the sample.Abbreviations are: PL, posterior limb; G, genu; AL, anterior limb; Tha, thalamus; Put, putamen; NC, caudate nucleus.


Fig. 3. Lateral part of the anterior limb (size 158.7×158.7 mm, DiI).The confocal image (a) and the same section in polarization mode (b)show parallel, horizontally running fibers which belong to the ante-rior thalamic peduncle. These fibers are traversed by single fiberswhich belong to the caudatopallidal system of fibers (a).

located at the lateral border of the first part of theposterior limb. A third fiber system is not organized inbundles. Single fibers run from the caudate nucleus toputamen and globus pallidus, thus these fibers traversethe anterior limb (Fig. 3). Isles of grey substance (theponticuli grisei) can be found in between the fibers ofthe anterior limb.

Fibers directed very steeply (almost 80–90° to theACPC-plane) run in the posterior part of the genu andin the anterior part of the posterior limb (area CI 3).

Fig. 4. Medial part of the anterior limb (size 158.7×158.7 mm, DiI),(a) confocal and (b) polarization. Two distinct systems of fibers canbe distinguished. The bundle of fibers in the middle represents fibersof the frontopontine tract. The horizontally cut fibers belong to theanterior thalamic peduncle. Because (b) is a summation effect overthe whole thickness of the sample (60 mm) the border between bothfiber systems is not as clear-cut as in (a).

anterior limb (area CI 1 and CI 2 in Fig. 8). The courseof a second fiber system is relatively steep. The fibers donot run into the thalamus, but pass the thalamus, andtheir slope is 60–80° in relation to the ACPC-plane.They are collected in bundles, which are organized inbands (Fig. 2). These steep bundles are intermingledwith the horizontal fiber bundles mentioned before(Fig. 4) and the angle between these bundles variesfrom 60 to 70°. The area of these two intermingled fibersystems (area CI 2 in Fig. 8) is located in the posteriortwo-thirds of the anterior limb. Its medial border corre-sponds to the tip of the genu and its posterior border is


Fig. 5. Anterior part of the posterior limb (size 158.7×158.7 mm, DiI), (a) and (b) confocal and (c) and (d) polarization. The bundles of steepfibers (transversely cut in the confocal images a,b) belong to the pyramidal tract. Horizontally cut fibers (b) belong to the superior thalamicpeduncle. Theses fibers can clearly be distinguished in the polarization mode (d).

The anterior border of this fiber system is the middlepart of the genu on the medial side. On the lateral sidethis fiber system begins at the edge of the putamen orthe globus pallidus more posteriorly and so this borderextends into the beginning of the posterior limb. Thesesteep fibers are intermingled with bundles of fiberswhich run horizontally and arise from the thalamus(Fig. 5). The number of these horizontal bundles de-creases continuously in the anterior–posteriordirection.

In the posterior part of the posterior limb the courseof the fibers is also steep, but they are not as homoge-

neously ordered as in the anterior part. Steep bundlesof fibers are oriented at slightly different angles and aretwisted. This produces an image of mosaic-like areas ofdifferent shades of grey in the polarization pictures(Fig. 6). In addition, fibers of large diameters like inarea CI 3 were not observed in such a high number(Tomasch, 1969). Bundles of horizontal fibers runningfrom the thalamus are sparse in this area CI 4. Theborder between area CI 3 and area CI 4 is located inthe posterior middle of the posterior limb. But thisborderline cannot be drawn very precisely as there is acontinuous transition between these fiber systems.


3.2. Interindi6idual differences

In all brains the parcellation of these fiber structureswas almost similar, but all fibers, especially in CI 3(Fig. 8), were more densely packed in smaller brainsthan in larger ones. A statistical analysis will follow. Inaddition, the border of this steep fiber system (area CI3) differed also slightly depending on the size of thebrain. In smaller specimen the anterior border of thesteep fibers was located more anteriorly in the genu.

Fig. 7. 3D-reconstruction of a confocal z-series. The picture illustratesthe steep, parallel fibers of the pyramidal tract.

Fig. 6. Posterior part of the posterior limb (size 158.7×158.7 mm,DiI). The fibers are twisted, so fibers with slightly different slopes canbe seen in the confocal image (a) and these appear as areas ofdifferent scales of grey in the polarization mode (b).

Fig. 8. Revised parcellation of the internal capsule in the threesections (S I, internal cerebral vein; S III, foramen interventriculareMonroi; S II, between I and III). We distinguish four major areaswhich differ in their fiber texture. These characteristic textures aredescribed in the text.

The posterior border differed most in all specimens, butthis border could not be defined clearly, because therewas a continuous transition of these systems of fibers.The ill-defined borders are marked as a dotted band inFig. 8.


4. Discussion

4.1. Methodological considerations

Traditionally the internal capsule is divided into theanterior limb, the genu and the posterior limb (Dejer-ine, 1901). The areas occupied by the distinct bundlesof fibers differ from the borders of the macroscopicallydefined parcellation of the internal capsule. In his clas-sical description, Vogt (1902) subdivided the posteriorlimb into three parts according to the intensity of theWeigert staining. This subdivision, however, does nottake care of the orientation of the fibers.

The central tracts of nervous fibers are important, forinstance, in stereotactic interventions focused on partic-ular tracts of fibers (e.g. in anterior capsulotomy).Many neurologic diseases affect the white matter of thebrain (e.g. multiple sclerosis, HIV) and result in neu-ropsychological impairments. Thus in many clinicalsettings more attention is presently paid to changes inthe white matter. The orientation of myelinated fiberscan be visualized by modern MRI procedures (diffusiontensor maps) (Douek et al., 1991; Peled et al., 1998).The technique presented here can be used to evaluatesuch results from an anatomical point of view.

Bundles of fibers are interesting if functional entitiesare investigated in the case of tracts of fibers. Changesin special tract systems can be found e.g. in amyo-trophic lateral sclerosis. In addition, right–left differ-ences may be detectable in some systems of fibers aswell as sex-dependent or age-related differences.

The new technical approach presented here can beused for mapping the 3D structure of central nervousfibers in the human brain. The method provides infor-mation not only about single nervous fibers but alsoabout bundles of fibers. Polarization microscopy is ableto differentiate distinct bundles of fibers and is a goodtool to describe the shape and size of these bundles.

Nevertheless, the slope of the fibers should provideinformation about the origin and the target of the

fibers. In ontogenesis, central nerve fibers will take theshortest way to reach their target (Maurach and Strian,1981). This shortest way could only be disturbed byanatomical structures which have to be avoided or byother systems of fibers. In the internal capsule the fibersoriginating in the frontal lobe have a different slopefrom fibers originating in the occipital lobe. Fibers fromcentral cortical regions proceed very steeply. On theother hand fibers travelling to the thalamus have toturn more horizontally to reach their thalamic targets,while fibers travelling to the pons follow their steeppath. Fig. 9 shows the different slopes of distinct fibersystems in the internal capsule. Difficulties in the differ-entiation of different tracts arise when fiber systemsshow only slightly different slopes as in the posteriorlimb (between area CI 3 and area CI 4), or whendistinct fiber systems may be mainly intermingled as inthe corona radiata.

The usefulness of confocal microscopy is rather obvi-ous. Single nerve fibers can clearly be visualized at highresolution, and confocal z-series provide three-dimen-sional information about the orientation of the nervefibers. DiI is a fluorescence marker which is often usedto investigate connectivity of nervous fibers (Honig andHume, 1986; Baker and Reese, 1993). The fact that thedye requires a relatively long time (3–5 months) fordiffusion is a disadvantage. We used this dye to label allmyelinated fibers in our sections and developed a pro-cedure which yields many samples quickly by stainingthe probes after sectioning. This way the method pro-vides the possibility to investigate a larger number ofspecimens in a shorter time.

The marker DiI labels myelin sheaths in particular.This way small unmyelinated fibers may not be imaged.They also do not contribute to the polarization images,since the anisotropy of the nervous tissue is the result ofradially orientated lipids in the myelin sheath. In thecentral nervous system, unmyelinated fibers were visual-ized accurately with electron microscopy (Keyserlingkand Schramm, 1984; Leenen et al., 1985; Aboitiz et al.,1992), showing for instance, some sex-dependent differ-ences in the ratio of myelinated to unmyelinated fibersto exist in the rat brain (Mack et al., 1995). Takingunmyelinated fibers into consideration may be veryinteresting for description of the organization of thevarious fiber tracts of our study, as there may existdifferences in the distribution of the amount of myelin-isation in the different tracts of fibers. This, however,was not possible to investigate here, but could form aninteresting field for future research.

The combination of confocal and polarized lighttechniques allows us to obtain detailed information ofthe fiber structure and orientation with confocal mi-croscopy and information about the localization andorientation of long myelinated fiber tracts with polar-ized light microscopy. Jouk et al. (1995) used both

Fig. 9. Different characteristic slopes of specific fiber systems: 1,anterior thalamic peduncle; 2, frontopontine tract; 3, superior thala-mic peduncle; 4, pyramidal tract; 5+6, parieto-occipto-pontine tract.


techniques for mapping the orientation of myocardialcells, but the use of the confocal microscope to produceboth confocal and polarized pictures of the same sec-tion at the same time is new.

We successfully demonstrated the architecture of thenervous fibers to influence the value of the impedancemeasurement at this point (Axer et al., 1999). Thusonline impedance measurements in a stereotactic inter-vention can be compared with the three-dimensionalstructure of nervous fibers in an atlas in order toperform a verification of the needle’s position.

Thus the method presented was developed to obtaindetailed information about the structural organizationof the myelinated fibers. However, the final identifica-tion of the various fiber tracts can only be done withknowledge of the localization of the different tractsystems. This knowledge is recorded in the literatureand will be discussed in detail. Each location in theinternal capsule contains a few fiber tracts and thesecan be differentiated according to their slope.

4.2. Corticopontine tracts

One of the major systems of fibers located in theinternal capsule is the corticopontine system of fibersprojecting to the pontine nuclei which in turn projectinto the cerebellum. Many investigations of this systemwere performed on monkeys. Most fibers of this systemwere demonstrated to arise from pericentral cortices(Dhanarajan et al., 1977; Brodal, 1978a,b, 1982;Wiesendanger et al., 1979; Hartmann-von Monakow etal., 1981). But also prefrontal (Leichnetz and Astruc,1976; Schahmann and Pandya, 1997), parieto-occipital(Schahmann and Pandya, 1992), cingulate (Vilenskyand Van Hoesen, 1981) and parahippocampal cortices(Schahmann and Pandya, 1993) project on the pontinenuclei.

In human beings, such results are relatively scarce(Tredici et al., 1990). The human corticopontine systemof fibers however comprises 20 times more fibers thanthe pyramidal tract system (Tomasch, 1969). In addi-tion, the association cortices in man are more devel-oped than in monkeys and may contribute tocerebrocerebellar circuits which also influence higherbehavioural functions (Leiner et al., 1991; Schahmann,1991).

The relatively steep bundles of fibers in the anteriorlimb (area CI 2), which pass the thalamus must befrontopontine fibers. The frontopontine tract (Arnold’sbundle) is a well known fiber tract in man (Meyers,1950). In post-mortem studies on degenerations in hu-man brains after prefrontal leucotomy, frontopontineas well as frontothalamic fibers were localized in theanterior limb of the internal capsule (Meyer, 1949;Beck, 1950). The frontopontine tract of fibers can alsobe visualized by the preparation method of Klingler

(Ludwig and Klingler, 1956 [Tabula 45, 47]). In studiesof monkey brains the frontopontine tract was alsolocalized in the anterior limb (Leichnetz and Astruc,1976).

We demonstrated the frontopontine tract not to be aseparate bundle in the anterior limb, as shown in classicanatomic textbooks (Kappers et al., 1967). Instead, itintermingles with the fiber bundles which come fromthe thalamus, and its border reaches into the lateralpart of the posterior limb. Earlier anatomical studiesusing Weigert myelin stains did not describe such fiberbundles (Vogt, 1902).

In addition, the fibers in the posterior part of theposterior limb (area CI 4) should be the parietooccipi-topontine projections because their course is not assteep as for the fibers from the pericentral cortex. Thissystem of fibers was localized in the posterior part ofthe posterior limb in the course of studies on degenera-tion (Dejerine, 1901), and by macroscopical dissection(Ludwig and Klingler, 1956 [Tabula 38]). Temporopon-tine fibers (Tuerck’s bundle) were not detected in thecourse of our sections because these fibers are locatedin more caudal planes (Dejerine, 1901).

In monkeys the parietooccipitopontine fibers are alsocollected in the posterior limb (Schahmann andPandya, 1992). The corticopontine fibers were distin-guishable as largely separate, but partially overlappingbundles (Schahmann and Pandya, 1992). This finding isvery similar to the picture we found in the posteriorlimb when using the polarization method (Fig. 6).

4.3. Pyramidal tract

Over decades the localization of the pyramidal tractin the internal capsule was a matter of dispute (David-off, 1990). The traditional concept of localization of thepyramidal tract in the anterior half of the posteriorlimb is based on the degeneration studies of Dejerine(1901). Analysis of lesions restricted to the internalcapsule using computer tomography (CT) demon-strated the localization of the pyramidal tract in theanterior half of the internal capsule and its topologicalorganization (Tredici et al. 1982; Bogousslavsky andRegli, 1990; Fries et al., 1993). In addition, it waspossible to reproduce this concept in defined experi-mental investigations in monkeys (Dawney and Glees,1986; Fries et al., 1993). Nevertheless, several otherwork groups located the pyramidal tract in the poste-rior half of the posterior limb in post-mortem casestudies (Englander et al., 1975; Hanaway and Young,1977), but also in a large number of exploratory stimu-lations of the internal capsule during stereotactic inter-ventions (Hardy et al., 1979) and during MRI-studieson amyotrophic lateral sclerosis (Yagishita et al., 1994).

This ambiguity was settled by Ross (1980) usingmacroscopical dissection techniques. The pyramidal


tract is at first located in the first half of the posteriorlimb but shifts posterior in more caudal sections, whichwas also demonstrated in myelogenic studies(Kretschmann, 1988). A detailed description of thesubcortical topography of the pyramidal tract was pro-vided by Ebeling and Reulen (1992).

In summary, the exact location of the pyramidal tractdepends on the level of section investigated, because thepyramidal tract features a change in its three-dimen-sional orientation (Maurach and Strian, 1981). In addi-tion, the orientation of the section is also crucial.Horizontal sections may vary up to 25°, and thesedifferences may change the relative location of the genuof the internal capsule. Our sections were cut parallel tothe ACPC line and this standardization to referencelandmarks allows all levels to be compared with eachother. Hardy et al. (1979) also used this referencesystem and analyzed a level 2 mm above the ACPCline. In contrast, our sections are located higher (4–8mm). Here, the pyramidal tract is located in thefirst half of the posterior limb (Ross, 1980;Kretschmann, 1988). The posterior shift of these fibersmust be expected at lower planes we did not actuallyinvestigate.

In our investigations we localized the pyramidal tractin area CI 3. The slope of the fibers located here is80–90°, and this has to be considered for thefibers running from pericentral cortices to the brainstem. We also found steep fibers located in the genuwhich apparently belong to corticobulbar fibers(Bogousslavsky and Regli, 1990). This localization ishowever not always found in all individuals. In smallerbrains these fibers are located more anteriorly than inlarger ones.

4.4. Thalamic radiation

Thalamocortical and corticothalamic fibers are oftenneglected in the description of the internal capsule ordepicted as separate bundles (Kappers et al., 1967).This is apparently due to the complex connectivity ofthe various thalamic nuclei (Axer and Niemann, 1994).Nevertheless, thalamic fibers intermingle diffusely withthe aforementioned fiber systems (Fries et al., 1993),but they can be differentiated, when they have to swinginto the thalamus.

The frontothalamic path consists of numerousbundles connecting the cortex of the frontal lobeto the medial and to a lesser extent to anterior thalamicnuclei (Hassler, 1982). This bundle of fibers con-verges in the anterior limb of the internal capsule(Kandel, 1989) and the great number of horizontallycut fibers in the anterior limb in our study (area CI 1and CI 2) belong to the anterior thalamic peduncle.The localization of these fibers can also be shownby means of the preparation method of Klingler (Lud-

wig and Klingler, 1956 [Tabula 54, 55]; Kandel, 1989)and was likewise confirmed by earlier degenerationstudies in the human brain (Dejerine, 1901; Beck,1950).

The projection of the lateral thalamic nuclei is mainlyto the pre- and postcentral gyri (Hassler, 1982). Thesefibers are located in the posterior limb of the internalcapsule (Kandel, 1989). We located the superior thala-mic peduncle in area CI 3 and CI 4 as also demon-strated by macroscopical preparation (Ludwig andKlingler, 1956 [Tabula 34, 35]). The thalamic bundlesof fibers decrease numerically in the anterior–posteriordirection of the posterior limb and they interminglediffusely with corticobulbar and corticospinal fibers(Kretschmann, 1988).

4.5. Caudatopallidal fibers

At least single fibers were found traversing the ante-rior limb and extending from the nucleus caudatus tothe nucleus lentiformis. In the monkey, anterogradelylabeled fibers arising from the caudate nucleus werefound, obliquely traversing the internal capsule andpenetrating the dorsal portion of the pallidum (Hazratiand Parent, 1992). These fibers traversing the anteriorlimb of the internal capsule have also been described inthe human brain (Hassler, 1974; Carpenter, 1982) andcould not be detected with conventional staining meth-ods (Dejerine, 1901; Vogt, 1902). In addition, thesefibers are also not detectable with polarizationmicroscopy.

4.6. The organization of the internal capsule

In summary, the organization of the internal capsuleas analyzed here differs from the classical models ofanatomic textbooks in some respects. The differenttracts of fibers are not individual, separate tracts offibers. Bundles of fibers intermingle with other bundles,but all have their characteristic orientation on theirown. The slope of the different systems of fiberschanges continuously in the anterior–posterior direc-tion of the internal capsule (Fig. 9). Thus differentbundles of fibers are distinguishable and so it is possibleto describe them separately. Using the 3D orientationof fibers as a criterion for parcellation as well as thedescription of bundles as a collection of fibers belong-ing to particular tracts results in a more function-re-lated description of the anatomy of central nervousfibers.

Acknowledgements

We would like to thank Petra Ibold and AnitaAgbedor for their technical assistance.


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Mapping of ﬁber orientation in human internal capsule by ... · The internal capsule was parcellated in the areas CI 1 to CI 4 containing ﬁbers of distinct orientation and structure,