Mapping of Fiber Orientation in Human Internal Capsule by Means Of

Home , Corticopontine fibers, Frontopontine fibers, Internal capsule

Journal of Neuroscience Methods 94 (2000) 165–175 www.elsevier.com/locate/jneumeth

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

Hubertus Axer *, Diedrich Graf v. Keyserlingk

Institut fu¨r Anatomie I, Uni6ersita¨tsklinikum 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 human cadaver brains were cut into comparable and standardized sections parallel to the ACPC-plane, stained with DiI, and analyzed using a combination of confocal and polarized light microscopy at the same time. This combination provides information about the structure and orientation of the fibers in great detail with confocal microscopy, and information about the localization and orientation of long myelinated fiber tracts with polarization microscopy. The internal capsule was parcellated in the areas CI 1 to CI 4 containing fibers of distinct orientation and structure, which enriches the macroscopically definable parcellation in the anterior and posterior limb. Fibers of the anterior thalamic peduncle intermingle with frontopontine tract fibers. Single fibers connect the caudate and the lentiform nucleus. The pyramidal tract is located in the anterior half of the posterior limb intermingled with fibers of the superior thalamic peduncle. Parietooccipitopontine fibers are located in the posterior part of the posterior limb. The slopes of the different systems of fibers change continuously in the anterior–posterior direction of the internal capsule. Using the 3D orientation of fibers as a criterion for parcellation, as well as the description of bundles as a collection of fibers belonging to particular tracts leads to a more function-related description of the anatomy of the internal capsule. The method can be used for interindividual, sex- or age-related comparisons of particular systems of fibers. © 2000 Elsevier Science B.V. All rights reserved.

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

1. Introduction of the needle’s position in a stereotactic procedure can be performed (Axer et al., 1998). In addition, the The fiber architecture especially of the white matter orientation of nervous fibers can be visualized by mod- in the human brain is presently the focus of interest. ern MRI procedures (Curnes et al., 1988; Douek et al., Many neurologic diseases affect the cerebral white mat- 1991; Peled et al., 1998). Nevertheless, a detailed ter, and modern imaging procedures such as magnetic anatomical mapping of fiber orientations in the adult resonance imaging (MRI) provide detailed visual infor- human brain is difficult to assess. Our aim was to mation about the white matter. Anatomical knowledge perform a mapping of the orientation of the fibers in about nervous fibers was achieved earlier by fiber the internal capsule. Thus we wanted to find out how preparations (Ebeling and Reulen, 1992), myelogenic anatomically defined fiber tracts are oriented in the studies (Kretschmann, 1988) and by observations after white matter. The method should give information brain injury (Fries et al., 1993). about fiber texture, fiber orientation and identification We were able to demonstrate the orientation and of specific fiber tracts. texture of the nervous fibers to influence the value of Therefore we used the combination of confocal laser impedance measurements, and so an online verification microscopy and polarized light microscopy. The confocal laser microscope allows information to be collected from well-defined optical sections. This is done by a * Corresponding author. Tel.: +49-241-8089100; fax: +49-241- 8888431. sequential illumination which is focused on one volume E-mail address: [email protected] (H. Axer) element of the specimen at a time (Wright et al., 1993).

This way stacks of optical sections can be produced, 2.2. Histologic preparation which allow three-dimensional reconstruction of the fiber texture and provide good information about the The slabs were first placed for 24 h in 10% sucrose orientation of the fibers at high resolution. solution and then in 30% sucrose solution until they Polarized light microscopy can selectively visualize sank to the bottom of the container. Afterwards the anisotropic structures. In polarization microscopy, opti- tissue was sectioned on a freezing microtome at 60 mm cally polarized light passes through a sample of tissue in the horizontal plane. This way all histologic sections and into a second polarizer (analyzer), which polarizes were orientated parallel to the ACPC-line. The sections light in a perpendicular plane with respect to the first were serially collected. Every second section was cover- polarizer. Birefringence is able to twist some of the light slipped without staining procedure. The other sections so that it can pass through the analyzer and be imaged. were kept in Tris buffer (0.1 M, pH 7.4) containing The birefringence of the nervous tissue has been well 0.1% Triton™ X-100 (Merck, Darmstadt, Germany) known for a long time (Schmidt, 1923, 1924; Schmitt for 24 h. Afterwards these sections were placed on and Bear, 1936; Kretschmann, 1967; Wolman, 1975). microscope slides and 3–4 drops of a DiI-solution (1 As the presence of anisotropy indicates polarity and mg DiI [1,1%-dilinoleyl-3,3,3%,3%-tetramethylindocarbo- order, polarization microscopy can be used to visualize cyanine perchlorate, FAST DiI™ oil, Molecular Probes long fiber tracts in the brain (Fraher and MacConnaill, Europe, Leiden, Netherlands] in 1100 ml dimethylsul- 1970; Miklossy and Van der Loos, 1991). The orienta- foxide [DMSO]) were put on these slides, and then the tion of the fibers influences the transmission of plane- slides were kept in a moist chamber at 37°C. DiI is polarized light at different velocities at different highly lipophilic and dissolves into lipid membranes. azimuths. This way the myelin sheaths of the nerve fibers in The combination of both techniques allowed us to particular are labeled. After 7 days, the sections were obtain detailed information of the fiber structure and mounted with the aqueous mounting medium Aqua- orientation with confocal microscopy and to collect tex™ (Merck, Darmstadt, Germany) and coverslipped. information about the localization and orientation of long myelinated fiber tracts with polarized light 2.3. Con6entional polarized light microscopy microscopy. The internal capsule in the adult human brain repre- The unstained sections were analyzed by conven- sents a collection of different systems of fibers closely tional polarized light microscopy. We used the Stemi located in a small space. It is a structure of high clinical SV 11 (Zeiss, Germany) with rotatable crossed polars importance, since the fibers of the pyramidal tract are for low magnification. This way we obtained a map of located here. Nevertheless the exact location of the the entire sample. A Leica DM IRBE (Leica Microsys- pyramidal tract in the internal capsule was a matter of tems, Heidelberg, Germany) microscope equipped with dispute for decades (Maurach and Strian, 1981). a rotatable stage and fixed crossed polars was used for higher magnification. The same microscope was used for confocal laser microscopy. 2. Materials and methods 2.1. Macroscopic preparation 2.4. Confocal laser scanning microscopy

Four human cadaver brains without macroscopically The DiI stained sections were analyzed with the definable pathology and without a history of neurologic Leica TCS NT confocal laser scanning microscope (Le- or psychiatric disease were fixed in 4% aqueous forma- ica Microsystems, Heidelberg, Germany). Since DiI lin solution and macroscopically prepared. The (maximum absorption: 549 nm, maximum emission: meninges were removed and the brains cut in the 564 nm) has fluorescent features similar to rhodamine median-sagittal plane. Important landmarks were iden- we used the standard rhodamine filter settings of the tified on the median surface of the hemispheres: the confocal microscope. anterior and posterior commissure, the interventricular foramen of Monro and the internal cerebral vein (or 2.5. Polarization with transmitted laser light the tela choroidea ventriculi tertii). The next two sections were cut parallel to the line between anterior and Polarized light microscopic pictures were produced posterior commissure (ACPC). The first horizontal sec- on a second channel of the confocal microscope. There- tion was made at the level of the internal cerebral vein. fore the transmission mode of the microscope was used. The second section was made at the level of the inter- In transmission mode the laser light passes through the ventricular foramen in the same direction. This way the sample and is detected in a photomultiplier at the other internal capsules in all eight hemispheres were sectioned side of the sample. Since laser light is polarized we had in comparable planes. to insert one polarization filter (analyzer) only into the H. Axer, D.G. Keyserlingk / Journal of Neuroscience Methods 94 (2000) 165–175 167

after rotation of 45°. The steeper these tracts of fibers are, the less bright the signal is. Thus different fiber tracts of different orientation and order produce areas of different brightness. In low magnification, the polarization picture provides information about the localization of different bundles of fibers (Fig. 2). At higher magnification single bundles of fibers can be detected. In polarization mode, the area resembling a bundle of fibers is a summation over the whole thickness of the section (60 mm), as the light must pass through the entire sample. At the same time confocal imaging allows the identification of single fibers and their texture (Figs. 3–6). The image produced with the confocal microscope is a small optical section in the sample. The angle of the fibers was defined by 3D-re- constructions from confocal z-series (Fig. 7).

Fig. 1. Combination of confocal and polarization microscopy. 3.1. Are6ised parcellation of the internal capsule path of the laser beam after the light has passed Fig. 8 shows the parcellation of the internal capsule through the sample. This way it was possible to analyze based on the distribution of differently oriented fibers. a single section with both confocal fluorescence and These areas are different from the borders of the polarization methods (Fig. 1) at the same time. macroscopically defined parcellation of the internal capsule (anterior limb, genu, posterior limb). The internal capsule was divided into four areas CI 1 to CI 4 for 3. Results a better orientation. Three distinct fiber systems can be distinguished in In polarization microscopy, the detectable signal de- the anterior limb. Many fibers originate from the thala- pends on the order and the inclination of the fibers. In mus and run in the direction of the anterior limb. In particular, parallel, horizontally cut fibers give a bright our sections these fibers are cut almost horizontally signal at a special azimuth whereas the signal decreases (Fig. 3) and are located in the whole area of the

Fig. 2. Anterior limb of the internal capsule as revealed by conventional polarization microscopy. Systems of ﬁbers of different orientation appear in 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. 168 H. Axer, D.G. Keyserlingk / Journal of Neuroscience Methods 94 (2000) 165–175

located at the lateral border of the first part of the posterior limb. A third fiber system is not organized in bundles. Single fibers run from the caudate nucleus to putamen and globus pallidus, thus these fibers traverse the anterior limb (Fig. 3). Isles of grey substance (the ponticuli grisei) can be found in between the fibers of the anterior limb. Fibers directed very steeply (almost 80–90° to the ACPC-plane) run in the posterior part of the genu and in the anterior part of the posterior limb (area CI 3).

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 anterior thalamic peduncle. These fibers are traversed by single fibers which belong to the caudatopallidal system of fibers (a). anterior limb (area CI 1 and CI 2 in Fig. 8). The course of a second fiber system is relatively steep. The fibers do not run into the thalamus, but pass the thalamus, and their slope is 60–80° in relation to the ACPC-plane. They are collected in bundles, which are organized in bands (Fig. 2). These steep bundles are intermingled with the horizontal fiber bundles mentioned before Fig. 4. Medial part of the anterior limb (size 158.7×158.7 mm, DiI), (Fig. 4) and the angle between these bundles varies (a) confocal and (b) polarization. Two distinct systems of fibers can from 60 to 70°. The area of these two intermingled fiber be distinguished. The bundle of fibers in the middle represents fibers of the frontopontine tract. The horizontally cut fibers belong to the systems (area CI 2 in Fig. 8) is located in the posterior anterior thalamic peduncle. Because (b) is a summation effect over two-thirds of the anterior limb. Its medial border corre- the whole thickness of the sample (60 mm) the border between both sponds to the tip of the genu and its posterior border is fiber systems is not as clear-cut as in (a). H. Axer, D.G. Keyserlingk / Journal of Neuroscience Methods 94 (2000) 165–175 169

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 steep fibers (transversely cut in the confocal images a,b) belong to the pyramidal tract. Horizontally cut fibers (b) belong to the superior thalamic peduncle. Theses fibers can clearly be distinguished in the polarization mode (d).

The anterior border of this fiber system is the middle neously ordered as in the anterior part. Steep bundles part of the genu on the medial side. On the lateral side of fibers are oriented at slightly different angles and are this fiber system begins at the edge of the putamen or twisted. This produces an image of mosaic-like areas of the globus pallidus more posteriorly and so this border different shades of grey in the polarization pictures extends into the beginning of the posterior limb. These (Fig. 6). In addition, fibers of large diameters like in steep fibers are intermingled with bundles of fibers area CI 3 were not observed in such a high number which run horizontally and arise from the thalamus (Tomasch, 1969). Bundles of horizontal fibers running (Fig. 5). The number of these horizontal bundles de- from the thalamus are sparse in this area CI 4. The creases continuously in the anterior–posterior border between area CI 3 and area CI 4 is located in direction. the posterior middle of the posterior limb. But this In the posterior part of the posterior limb the course borderline cannot be drawn very precisely as there is a of the fibers is also steep, but they are not as homoge- continuous transition between these fiber systems. 170 H. Axer, D.G. Keyserlingk / Journal of Neuroscience Methods 94 (2000) 165–175

3.2. Interindi6idual differences

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

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

Fig. 8. Revised parcellation of the internal capsule in the three sections (S I, internal cerebral vein; S III, foramen interventriculare Monroi; S II, between I and III). We distinguish four major areas which differ in their ﬁber texture. These characteristic textures are described in the text.

The posterior border differed most in all specimens, but this border could not be defined clearly, because there 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 can was a continuous transition of these systems of fibers. be seen in the confocal image (a) and these appear as areas of The ill-defined borders are marked as a dotted band in different scales of grey in the polarization mode (b). Fig. 8. H. Axer, D.G. Keyserlingk / Journal of Neuroscience Methods 94 (2000) 165–175 171

4. Discussion fibers. In ontogenesis, central nerve fibers will take the shortest way to reach their target (Maurach and Strian, 4.1. Methodological considerations 1981). This shortest way could only be disturbed by anatomical structures which have to be avoided or by Traditionally the internal capsule is divided into the other systems of fibers. In the internal capsule the fibers anterior limb, the genu and the posterior limb (Dejer- originating in the frontal lobe have a different slope ine, 1901). The areas occupied by the distinct bundles from fibers originating in the occipital lobe. Fibers from of fibers differ from the borders of the macroscopically central cortical regions proceed very steeply. On the defined parcellation of the internal capsule. In his clas- other hand fibers travelling to the thalamus have to sical description, Vogt (1902) subdivided the posterior turn more horizontally to reach their thalamic targets, limb into three parts according to the intensity of the while fibers travelling to the pons follow their steep Weigert staining. This subdivision, however, does not path. Fig. 9 shows the different slopes of distinct fiber take care of the orientation of the fibers. systems in the internal capsule. Difficulties in the differ- The central tracts of nervous fibers are important, for entiation of different tracts arise when fiber systems instance, in stereotactic interventions focused on partic- show only slightly different slopes as in the posterior ular tracts of fibers (e.g. in anterior capsulotomy). limb (between area CI 3 and area CI 4), or when Many neurologic diseases affect the white matter of the distinct fiber systems may be mainly intermingled as in brain (e.g. multiple sclerosis, HIV) and result in neu- the corona radiata. ropsychological impairments. Thus in many clinical The usefulness of confocal microscopy is rather obvi- settings more attention is presently paid to changes in ous. Single nerve fibers can clearly be visualized at high the white matter. The orientation of myelinated fibers resolution, and confocal z-series provide three-dimen- can be visualized by modern MRI procedures (diffusion sional information about the orientation of the nerve tensor maps) (Douek et al., 1991; Peled et al., 1998). fibers. DiI is a fluorescence marker which is often used The technique presented here can be used to evaluate to investigate connectivity of nervous fibers (Honig and such results from an anatomical point of view. Hume, 1986; Baker and Reese, 1993). The fact that the Bundles of fibers are interesting if functional entities dye requires a relatively long time (3–5 months) for are investigated in the case of tracts of fibers. Changes diffusion is a disadvantage. We used this dye to label all in special tract systems can be found e.g. in amyo- myelinated fibers in our sections and developed a pro- trophic lateral sclerosis. In addition, right–left differ- cedure which yields many samples quickly by staining ences may be detectable in some systems of fibers as the probes after sectioning. This way the method pro- well as sex-dependent or age-related differences. vides the possibility to investigate a larger number of The new technical approach presented here can be specimens in a shorter time. used for mapping the 3D structure of central nervous The marker DiI labels myelin sheaths in particular. fibers in the human brain. The method provides infor- This way small unmyelinated fibers may not be imaged. mation not only about single nervous fibers but also They also do not contribute to the polarization images, about bundles of fibers. Polarization microscopy is able since the anisotropy of the nervous tissue is the result of to differentiate distinct bundles of fibers and is a good radially orientated lipids in the myelin sheath. In the tool to describe the shape and size of these bundles. central nervous system, unmyelinated fibers were visual- Nevertheless, the slope of the fibers should provide ized accurately with electron microscopy (Keyserlingk information about the origin and the target of the and Schramm, 1984; Leenen et al., 1985; Aboitiz et al., 1992), showing for instance, some sex-dependent differences in the ratio of myelinated to unmyelinated fibers to exist in the rat brain (Mack et al., 1995). Taking unmyelinated fibers into consideration may be very interesting for description of the organization of the various fiber tracts of our study, as there may exist differences 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 an interesting field for future research. The combination of confocal and polarized light techniques allows us to obtain detailed information of the fiber structure and orientation with confocal mi- Fig. 9. Different characteristic slopes of specific fiber systems: 1, croscopy and information about the localization and anterior thalamic peduncle; 2, frontopontine tract; 3, superior thala- orientation of long myelinated fiber tracts with polar- mic peduncle; 4, pyramidal tract; 5+6, parieto-occipto-pontine tract. ized light microscopy. Jouk et al. (1995) used both 172 H. Axer, D.G. Keyserlingk / Journal of Neuroscience Methods 94 (2000) 165–175 techniques for mapping the orientation of myocardial (Ludwig and Klingler, 1956 [Tabula 45, 47]). In studies cells, but the use of the confocal microscope to produce of monkey brains the frontopontine tract was also both confocal and polarized pictures of the same sec- localized in the anterior limb (Leichnetz and Astruc, tion at the same time is new. 1976). We successfully demonstrated the architecture of the We demonstrated the frontopontine tract not to be a nervous fibers to influence the value of the impedance separate bundle in the anterior limb, as shown in classic measurement at this point (Axer et al., 1999). Thus anatomic textbooks (Kappers et al., 1967). Instead, it online impedance measurements in a stereotactic inter- intermingles with the fiber bundles which come from vention can be compared with the three-dimensional the thalamus, and its border reaches into the lateral structure of nervous fibers in an atlas in order to part of the posterior limb. Earlier anatomical studies perform a verification of the needle’s position. using Weigert myelin stains did not describe such fiber Thus the method presented was developed to obtain bundles (Vogt, 1902). detailed information about the structural organization In addition, the fibers in the posterior part of the of the myelinated fibers. However, the final identifica- posterior limb (area CI 4) should be the parietooccipi- tion of the various fiber tracts can only be done with topontine projections because their course is not as knowledge of the localization of the different tract steep as for the fibers from the pericentral cortex. This systems. This knowledge is recorded in the literature system of fibers was localized in the posterior part of and will be discussed in detail. Each location in the the posterior limb in the course of studies on degenera- internal capsule contains a few fiber tracts and these tion (Dejerine, 1901), and by macroscopical dissection can be differentiated according to their slope. (Ludwig and Klingler, 1956 [Tabula 38]). Temporopon- tine fibers (Tuerck’s bundle) were not detected in the 4.2. Corticopontine tracts course of our sections because these fibers are located in more caudal planes (Dejerine, 1901). One of the major systems of fibers located in the In monkeys the parietooccipitopontine fibers are also internal capsule is the corticopontine system of fibers collected in the posterior limb (Schahmann and projecting to the pontine nuclei which in turn project Pandya, 1992). The corticopontine fibers were distin- into the cerebellum. Many investigations of this system guishable as largely separate, but partially overlapping were performed on monkeys. Most fibers of this system bundles (Schahmann and Pandya, 1992). This finding is were demonstrated to arise from pericentral cortices very similar to the picture we found in the posterior (Dhanarajan et al., 1977; Brodal, 1978a,b, 1982; limb when using the polarization method (Fig. 6). Wiesendanger et al., 1979; Hartmann-von Monakow et al., 1981). But also prefrontal (Leichnetz and Astruc, 4.3. Pyramidal tract 1976; Schahmann and Pandya, 1997), parieto-occipital (Schahmann and Pandya, 1992), cingulate (Vilensky Over decades the localization of the pyramidal tract and Van Hoesen, 1981) and parahippocampal cortices in the internal capsule was a matter of dispute (David- (Schahmann and Pandya, 1993) project on the pontine off, 1990). The traditional concept of localization of the nuclei. pyramidal tract in the anterior half of the posterior In human beings, such results are relatively scarce limb is based on the degeneration studies of Dejerine (Tredici et al., 1990). The human corticopontine system (1901). Analysis of lesions restricted to the internal of fibers however comprises 20 times more fibers than capsule using computer tomography (CT) demon- the pyramidal tract system (Tomasch, 1969). In addi- strated the localization of the pyramidal tract in the tion, the association cortices in man are more devel- anterior half of the internal capsule and its topological oped than in monkeys and may contribute to organization (Tredici et al. 1982; Bogousslavsky and cerebrocerebellar circuits which also influence higher Regli, 1990; Fries et al., 1993). In addition, it was behavioural functions (Leiner et al., 1991; Schahmann, possible to reproduce this concept in defined experi- 1991). mental investigations in monkeys (Dawney and Glees, The relatively steep bundles of fibers in the anterior 1986; Fries et al., 1993). Nevertheless, several other limb (area CI 2), which pass the thalamus must be work groups located the pyramidal tract in the poste- frontopontine fibers. The frontopontine tract (Arnold’s rior half of the posterior limb in post-mortem case bundle) is a well known fiber tract in man (Meyers, studies (Englander et al., 1975; Hanaway and Young, 1950). In post-mortem studies on degenerations in hu- 1977), but also in a large number of exploratory stimu- man brains after prefrontal leucotomy, frontopontine lations of the internal capsule during stereotactic inter- as well as frontothalamic fibers were localized in the ventions (Hardy et al., 1979) and during MRI-studies anterior limb of the internal capsule (Meyer, 1949; on amyotrophic lateral sclerosis (Yagishita et al., 1994). Beck, 1950). The frontopontine tract of fibers can also This ambiguity was settled by Ross (1980) using be visualized by the preparation method of Klingler macroscopical dissection techniques. The pyramidal H. Axer, D.G. Keyserlingk / Journal of Neuroscience Methods 94 (2000) 165–175 173 tract is at first located in the first half of the posterior wig and Klingler, 1956 [Tabula 54, 55]; Kandel, 1989) limb but shifts posterior in more caudal sections, which and was likewise confirmed by earlier degeneration was also demonstrated in myelogenic studies studies in the human brain (Dejerine, 1901; Beck, (Kretschmann, 1988). A detailed description of the 1950). subcortical topography of the pyramidal tract was pro- The projection of the lateral thalamic nuclei is mainly vided by Ebeling and Reulen (1992). to the pre- and postcentral gyri (Hassler, 1982). These In summary, the exact location of the pyramidal tract fibers are located in the posterior limb of the internal depends on the level of section investigated, because the capsule (Kandel, 1989). We located the superior thala- pyramidal tract features a change in its three-dimen- mic peduncle in area CI 3 and CI 4 as also demon- sional orientation (Maurach and Strian, 1981). In addi- strated by macroscopical preparation (Ludwig and tion, the orientation of the section is also crucial. Klingler, 1956 [Tabula 34, 35]). The thalamic bundles Horizontal sections may vary up to 25°, and these of fibers decrease numerically in the anterior–posterior differences may change the relative location of the genu direction of the posterior limb and they intermingle of the internal capsule. Our sections were cut parallel to diffusely with corticobulbar and corticospinal fibers the ACPC line and this standardization to reference (Kretschmann, 1988). landmarks allows all levels to be compared with each other. Hardy et al. (1979) also used this reference 4.5. Caudatopallidal fibers system and analyzed a level 2 mm above the ACPC line. In contrast, our sections are located higher (4–8 At least single fibers were found traversing the ante- mm). Here, the pyramidal tract is located in the rior limb and extending from the nucleus caudatus to first half of the posterior limb (Ross, 1980; the nucleus lentiformis. In the monkey, anterogradely Kretschmann, 1988). The posterior shift of these fibers labeled fibers arising from the caudate nucleus were must be expected at lower planes we did not actually found, obliquely traversing the internal capsule and investigate. penetrating the dorsal portion of the pallidum (Hazrati In our investigations we localized the pyramidal tract and Parent, 1992). These fibers traversing the anterior in area CI 3. The slope of the fibers located here is limb of the internal capsule have also been described in 80–90°, and this has to be considered for the the human brain (Hassler, 1974; Carpenter, 1982) and fibers running from pericentral cortices to the brain could not be detected with conventional staining meth- stem. We also found steep fibers located in the genu ods (Dejerine, 1901; Vogt, 1902). In addition, these which apparently belong to corticobulbar fibers fibers are also not detectable with polarization (Bogousslavsky and Regli, 1990). This localization is microscopy. however not always found in all individuals. In smaller brains these fibers are located more anteriorly than in 4.6. The organization of the internal capsule larger ones. In summary, the organization of the internal capsule 4.4. Thalamic radiation as analyzed here differs from the classical models of anatomic textbooks in some respects. The different Thalamocortical and corticothalamic fibers are often tracts of fibers are not individual, separate tracts of neglected in the description of the internal capsule or fibers. Bundles of fibers intermingle with other bundles, depicted as separate bundles (Kappers et al., 1967). but all have their characteristic orientation on their This is apparently due to the complex connectivity of own. The slope of the different systems of fibers the various thalamic nuclei (Axer and Niemann, 1994). changes continuously in the anterior–posterior direc- Nevertheless, thalamic fibers intermingle diffusely with tion of the internal capsule (Fig. 9). Thus different the aforementioned fiber systems (Fries et al., 1993), bundles of fibers are distinguishable and so it is possible but they can be differentiated, when they have to swing to describe them separately. Using the 3D orientation into the thalamus. of fibers as a criterion for parcellation as well as the The frontothalamic path consists of numerous description of bundles as a collection of fibers belong- bundles connecting the cortex of the frontal lobe ing to particular tracts results in a more function-re- to the medial and to a lesser extent to anterior thalamic lated description of the anatomy of central nervous nuclei (Hassler, 1982). This bundle of fibers con- fibers. verges in the anterior limb of the internal capsule (Kandel, 1989) and the great number of horizontally cut fibers in the anterior limb in our study (area CI 1 Acknowledgements and CI 2) belong to the anterior thalamic peduncle. The localization of these fibers can also be shown We would like to thank Petra Ibold and Anita by means of the preparation method of Klingler (Lud- Agbedor for their technical assistance. 174 H. Axer, D.G. Keyserlingk / Journal of Neuroscience Methods 94 (2000) 165–175

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