Abstract We estimated the total neurone number, glialnumber, and glial index (ratio glial cells/neurone) in thethalamic mediodorsal nucleus (MD) in seven patients suf-fering from Huntington’s disease (HD; four males, threefemales, mean age 52.4 ± 13.6 years) and age- and sex-matched controls (four males, three females, mean age53.6 ± 12.1 years) by means of a stereological protocol.The mean total neurone number (NT̄) in the MD of con-trols was 2,985,188 ± 174,710, the mean glial number(GT̄; astrocytes, oligodendrocytes) 21,785,008 ± 2,986,678,and the glial index 7.29 ± 0.88. In HD, the average neu-rone number was decreased by 23.8% to 2,275,321 ±247,162 (Mann-Whitney U-test P < 0.05), the mean glialnumber by 29.7 % to 15,318,895 ± 1,722,524 (Mann-Whitney U-test P < 0.05), the glial index was slightly re-duced to 6.81 ± 1.06. Gallyas’ impregnation for thedemonstration of fibrous astroglia gave strongly positive

results in all cases with HD and negative results in thecontrols. The morpho-functional correlation of the resultsis complicated because individual variability, presence ofsegregated and parallel neuronal circuits, and plasticity ofthe adult human CNS must be considered.

Key words Human brain · Thalamus · Myeloarchitectonic · Nerve cell number · Optical disector

Introduction

The mediodorsal nucleus (MD) is a major component ofthe thalamic nuclear complex surrounded, in the main, bythe internal medullary lamina. Its medial border is madeup by the third ventricle and by small midline nuclei thatare interspersed between the ventricular wall and the me-dial part of the MD. Olszewski [77] subdivided the MD of rhesus monkey into a magnocellular, a parvocellular,multiform, and a densocellular subnucleus. Hassler [44](monkey, man) and Hirai and Jones [51] (man) establishedfour subdivisions in the primate MD but used differentterminology (Hassler: medialis fibrosus, medialis fascicu-losus, medialis caudalis, paralamellaris; Hirai and Jones:medial magnocellularis, dorsolateral parvocellularis, ven-tral multiformis, part of the centralis lateralis). The MD ofprimates is reciprocally connected with the frontal cortex[85, 88, 104]. The absolute and relative increase in MDvolume of primates and, in particular, in man has been at-tributed to a considerable increase in size of the prefrontalcortex in the phylogenetic scale [5, 6, 32, 33, 52, 75, 83].

Corticothalamic connections in humans have been investigated after traumatic, vascular, or tumour lesionswith subsequent neuronal degeneration. Neuropathologi-cal investigations of the diseased human brain confirmedreciprocal connections of the MD with the prefrontal cor-tex [3, 11, 31, 39, 45, 67, 71, 94, 104]. In a previous study[48] we observed an average 33% neuronal loss in thecerebral cortex of patients with Huntington’s disease(HD). An attenuation of lamina VI and nerve cell loss inlamina V, which contain thalamic projection neurones

Helmut Heinsen · Udo Rüb · Manfred Bauer ·Gerd Ulmar · Birgitt Bethke · Michael Schüler ·Felix Böcker · Wolfgang Eisenmenger · Monika Götz ·Hubert Korr · Christoph Schmitz

Nerve cell loss in the thalamic mediodorsal nucleus in Huntington’s disease

Acta Neuropathol (1999) 97 :613–622 © Springer-Verlag 1999

Received: 9 September 1997 / Revised: 24 August, 12 November 1998 / Accepted: 13 November 1998

REGULAR PAPER

H. Heinsen (Y) · U. Rüb1

Morphologische Hirnforschung der Psychiatrischen Klinik, Josef-Schneider-Str. 2, D-97080 Würzburg, Germanye-mail: heinsen@mail.uni-wuerzburg.de, Tel.: +49-931-2017550, Fax: +49-931-960498

M. Bauer · G. UlmarPsychiatrisches Landeskrankenhaus, D-69155 Wiesloch, Germany

B. BethkeInstitut für Pathologie, D-95445 Bayreuth, Germany

M. Schüler · F. BöckerNervenkrankenhaus des Bezirks Oberfranken, D-95445 Bayreuth, Germany

W. EisenmengerInstitut für Rechtsmedizin der Universität, D-80337 München, Germany

M. GötzInstitut für Pathologie, D-63739 Aschaffenburg, Germany

H. Korr · C. SchmitzInstitut für Anatomie der RWTH, D-52057 Aachen, Germany

Present address:1 Zentrum der Morphologie, Johann-Wolfgang-Goethe-Universität,Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany

suggests retrograde neurone loss in HD patients [48, 96,97]. We have, therefore, utilised a stereological method toinvestigate a hypothesised neurone loss in the thalamicMD in patients with HD.

Materials and methods

The left hemispheres of seven terminal stages of HD patients(three females, four males, mean age 52.4 ± 13.6 years) and sevenleft hemispheres of age- and sex-matched controls (three females,four males, mean age 53.6 ± 12.1 years) without known neuropsy-chiatric diagnoses were investigated. Autopsy was performed onlyafter consent of the relatives of the deceased according to the lawsof the Federal Republic of Germany. Clinical pictures and courseof HD in patients have been summarised in previous publications[48,49]. The investigation of the brains was approved by the Ethi-cal Board of the Faculty of Medicine at the University of Würz-burg.

The hemispheres were immersion-fixed in 10% formalin (1 partof concentrated solution of formalin diluted with 9 parts of water)for at least 3 months, embedded in gelatine, and then deep-frozen.The entire hemisphere was serially cut at 700 µm thickness start-ing at the frontal pole of the hemisphere with a random startingpoint within the first 700 µm to fulfil the requirements for system-atic random sampling (see e.g. [41, 112]). The sections were thendistributed in three parallel series, one was stained by gallocyanin[46] and used for counting neurones and glial cells (see also [46,48, 49]), another for new samplings, the last for paraffin embed-ding. Paraffin-embedded tissue was cut with a microtome at 10–14µm, and stained with van Gieson stain, hematoxylin-eosin, andGallyas’ [34] silver impregnation to reveal fibrous astrogliosis.

Outlines and borders of the thalamic MD in these stained andmounted serial sections were identified using a wide-field stereo-microscope at × 7.5 magnification. The depth of focus is low usinga steromicroscope which gives a three-dimensional impression of thalamic nuclear arrangement in thick gallocyanin-stained sec-tions. Single stacked thalamic neurones can be distinguished dif-fering by less than 0.1 mm in the optical plane. Thus, the depth offocus was far lower than in the Figs. 1 and 2, which were taken bya single lens reflex camera. Area estimation of the MD in each section was performed at the same magnification by means of a 10 × 10-mm ocular grid subdivided by 20 × 20 lines. All crossingpoints (P) of this grid hitting the MD were counted. The averagesection thickness (th––) of the stained and mounted sections was de-termined with the fine adjustment knob of an Olympus BH micro-scope [49]. Estimated total MD volumes (V̂MD) were obtained us-ing Cavalieri’s principle [41, 109] according to:

(1)

Where ap is the area associated with one crossing point in the gridat final magnification (ap = 2.97566 mm2), ds is the distance of thesections (ds = 3 since every third section was analysed), Pi is thesum of crossing points counted for section i, and ns is the numberof equidistant sections through the MD (ns = 8–10). Average neu-rone and glial density was estimated at ×400 magnification (oil-immersion objective 40/1.0 combined with ×10 wide-field eye-pieces) using optical disectors as described in the literature (see,e.g., [21, 40–42, 66, 100, 110]). The mounted sections were fixedon the mechanical stage of an Olympus BH microscope and weremoved in 0.865-mm intervals in both directions X and Y as de-scribed previously [49], beginning at the upper pole of the oval-shaped MD. A 5 × 5-mm ocular grid subdivided by 10 × 10 lineswas used as an unbiased counting frame. For neurone counts (NC)all 100 fields of this ocular grid were used, resulting in an area ofoptical disectors (aOD – NC) of 15,625 µm2 (see also [47]). All nucleoli of neurones coming anew into view in a depth between 20 µm and 49.7 µm were counted. Thus, the height of the opticaldisectors (hOD – NC) was 29.7 µm. The movement in the Z axis

(height of the optical disectors) was controlled mechanically byone vertical and two horizontal metal pins attached to the fine andcoarse adjustment knob of the microscope [47]. Counting of nu-cleoli was legitimated by the observation that the overwhelmingmajority of neurones contained only one nucleolus.

Estimated average neurone density per volume wascalculated according to:

(2)

Where nx is the number of optical disectors hitting the MD and nythe number of optical disectors the midpoint of which was foundinside the MD (see also [101]). Estimated total numbers of neu-rones (N̂NC) were obtained with:

(3)

For glial counts (GC) only 16 fields of the ocular grid were used.Thus, (aOD – GC) was 2,500 µm2. All nuclei of glial cells (oligoden-droglia, astroglia, extremely rare presumptive microglia) cominganew into view in a depth between 20 µm and 49.7 µm werecounted. Thus, the height of the optical disectors (hOD – GC) was also29.7 µm. Using Nissl-stained sections it was not possible to distin-guish unequivocally between astroglial, oligodendroglial, or micro-glial nuclei. Estimated average glial density per unit volume

and estimated total number of glia cells (N̂GC) were cal-culated similar to formulae (2) and (3).

Volume estimation, neurone, and glial cell number countingwas performed by U.R. In a pilot study on five control cases wefound a statistically significant inter-rater reliability in neurone es-timation between H.H. and UR. HD cases and control cases couldnot be investigated “blind” to the diagnosis because striatal atro-phy was already macroscopically visible.

Summarising, approximately 121 points (73–158), approxi-mately 1,010 neurones (763–1,288), and approximately 1,130 glialcells (708–1,723) were counted per MD using this estimation pro-cedure. Numbers of neurones and glial cells counted were farhigher than currently performed in stereological studies compara-ble with that carried out here [49, 78, 79, 84, 86, 101, 112–114]and far higher than currently outlined in recent papers describing the theoretical background of disector stereology [20, 21, 42, 66,110, 111]. For the theoretical background see the accompanyingpaper of Schmitz et al. [91]. In a pilot experiment the total numberof neurones of control case C3 was estimated 15 times using dis-ectors with an area of 5,625 µm2 and a height of 29.7 µm, but withan X-Y-distance of 1,200 µm. This estimation procedure resultedon average in counting 161 neurones per repetition. Glial indices(I) were calculated for each MD according to:

(4)

Differences in neurone number, glial number, and glial index be-tween controls and HD cases were analysed by the Mann-WhitneyU-test [15].

Some representative gallocyanin-stained sections were pho-tographed with a 24 × 36-mm single lens reflex camera on AgfaAPX 25 black and white negative film. The negatives werescanned with a Polaroid Sprint Scan scanner and the scanned TIFfiles were processed on a PC with Micrografx PicturePublisher. Ifnecessary, contrast and tone scale were modified to generate a uni-form tone. Superficially adherent gelatine was excluded electroni-cally and the background was filled with 7% grey tone or 95%grey tone (see Figs. 1 and 4). Thereafter, the TIF files were im-ported into Micrografx Designer and numbers, letters, and desig-nations were added. The Designer files were converted into post-script files and exposed on photographic paper by a Linotronic 330printing machine (152 lpi).

IN

NGC

GC

NC

=ˆ

ˆ

N̂V – GC( )

ˆ ˆ ˆN V NNC MD V – NC= ×

N̂

NC

ny h aV – NC

jj =1

nx

od od

=( )

× ×

∑

N̂V – NC( )

V̂ th a d PMD p s ii =1

ns

= × × × ∑

614

Results

Microscopical delineation of the MD

The nerve cell density in most parts of the human brain isrelatively low compared to that of the brain of small ro-

dents. We could, therefore, investigate unusually thickgallocyanin-stained serial sections. In our experience,staining procedures, dehydration, and mounting of thestained sections results in considerable shrinkage of braintissue. Original section thickness of frozen tissue can de-crease by about 20% from 700 to 560 µm. Investigation of

615

these transparent 500- to 600-µm-thick sections throughthe human brain with the aid of a stereomicroscope givesan appreciation of the complicated three-dimensionalarrangement of individual neurones into nuclei and lami-nae. It was, therefore, possible to delineate unequivocallythe MD border in all sections investigated through the ex-tended thalamic nucleus (Figs. 1, 3). Using dark-field illu-mination, fibres and lamellae within the thalamic complexbecame visible in our gallocyanin-stained sections. Wecould additionally use these myeloarchitectonic criteria todelineate the MD from adjacent nuclei (Figs. 2, 4). Sub-nuclei similar to those described by a number of authorscould be recognised, but the mediolateral borders betweenthese MD subnuclei were less sharp than the external MDborder. However, rostrocaudal transitions between MDsubnuclei were extremely difficult to delineate withinclosely spaced sections, thus morphometry of MD subnu-clei was not possible in our material.

We observed no consistent morphological changes inMD of HD cases with low-power microscopy. The MDappeared smaller, clustering of neurones within MD wasless marked, and fibre density was increased within theMD of HD cases (Figs. 3, 4). However, these criteria wereinsufficient for a diagnosis of HD.

Light microscopy of gallocyanin- and Gallyas-stained slices of MD

We were unable to find consistent qualitative cytologicalneuronal changes in the MD using gallocyanin staining(Figs. 5, 6). In contrast to this, however, we always observedfibrous astrogliosis in HD cases after embedding small tis-sue sections from parallel thick frozen sections into paraffinand staining with Gallyas’ [34] impregnation (Fig. 7). Withthe exception of some perivascular coarse fibres and ery-throcytes all other tissue components of paraffin-embeddedsections through the MD of the controls remained unstained.

Quantitative results

The estimated MD volume in the HD was cases on average25.3% smaller than in the control cases (451 ± 55.5 mm3

vs 604 ± 62.6 mm3; mean ± SD; for details see Table 1).The nerve cell density was in the HD cases on average1.7% greater than in the control cases (5,053 vs 4,968 neu-rones/mm3). However, the total neurone number was inthe HD cases reduced by 23.8% compared with the con-trol cases (2,275,321 ± 247,162 vs 2,985,188 ± 174,710).This difference of 23.8% was statistically significant (P <0.05, Mann-Whitney U-test). The total number of glial cellsin the MD was also greater in control cases than in HD ca-ses (21,785,008 ± 2,986,678 vs. 15,318,895 ± 1,722,524;P < 0.05, Mann-Whitney U-test). The glial index wasslightly smaller in HD cases (6.81 ± 1.06) than in the con-trol cases (7.29 ± 0.88). These differences were not statis-tically significant.

Discussion

Possible mechanisms of neuronal degeneration in MD

The most prominent neuropathological finding in HD isbilateral atrophy of the striatum. Neuronal degenerationmainly affects striatal GABA-ergic medium spiny stellatecells [38]. Large cholinergic [58] and somatostatin-con-taining NPY-positive neurones [24, 27] are spared. Striatalnerve cell loss parallels the clinical severity in HD [73,107]. Quantitative investigations during the last two de-cades have shown that nerve cell loss is not confined tothe striatum but is also evident in cortical as well as sub-cortical grisea including the thalamic centromedian-para-fascicular complex (–55% [49]), the globus pallidus(–40% [58]), the cerebral cortex (–33% [48]), the sub-thalamic nucleus (–25% [58]), and, in this study, the thal-amic mediodorsal nucleus (–23.8%; Table 1). The quan-tity of nerve cell loss in affected grey matter does notnecessarily reflect the chronological sequence of neu-ronal degeneration although a majority of investigatorsassumes that the striatum is the first structure to exhibitneurone degeneration. This missing evidence betweenquantitative neurone loss and chronological progressionmakes insights into the pathogenesis of HD difficult and

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Fig.1 Gallocyanin-stained section through central parts of MD intransparent illumination; control female, 58-year-old

Fig.2 Same section as in Fig. 1, darkfield illumination

Fig.3 Gallocyanin-stained section from a 54-year-old female HDpatient

Fig.4 Same section as in Fig. 3, darkfield illumination. Plane ofsection in Figs. 3, 4 is slightly more caudal than in Figs. 1, 2

Figs.1–4 All sections cut in the coronal plane. Terminology indarkfield illumination according to Hassler [45] as contrasted withAmerican terminology in Figs. 1, 3 according to Walker [108]. Forabbreviations see list. The internal medullary lamina which sepa-rates lateral parts of the MD from adjacent dorsal and lateral thal-amic nuclear complexes and which envelopes the intralaminar nu-clei Cl (i.La.ip.) and the centromedian-parafascicular complex(CM-Pf) becomes visible after darkfield illumination (Figs. 2 and4) of the same section. This greatly facilitated the delineation ofthe MD. (Readers interested in individual variability of thalamiccytoarchitectonics are referred to Dewulf [26]. American syn-onyms: AD Ncl. antero-dorsalis, AV Ncl. antero-ventralis, Cl Ncl.centralis lateralis, CM Ncl. centrum medianum, LP Ncl. lateralisposterior, MDmc Ncl. mediodorsalis pars magnocellularis, MDpcNcl. mediodorsalis pars parvicellularis, Pf Ncl. parafascicularis,Pm Ncl. paramedianus, Pt Ncl. parataenialis, Re Ncl. reuniens,VIM Ncl. ventrointermedius, VL Ncl. ventrolateralis, VPLo Ncl.ventralis posterolateralis, VPM Ncl. ventralis posteromedialis. Ter-minology of Hassler [45] A.d. Ncl. antero-dorsalis, A.pr. Ncl. an-tero-principalis, Cemc Ncl. centralis magnocellularis, Cepc Ncl.centralis parvocellularis, Cu Ncl. cucullaris, D.o. Ncl. dorsalisoralis, M.fa. Ncl. medialis fasciculosus, M.fa.s. Ncl. fasciculosussuperior, M.fi. Ncl. medialis fibrosus, Pt Ncl. parataenialis, St.mdStria medullaris thalami, V.c.a.e. Ncl. ventrocaudalis anterior ex-ternus, V.c.a.i Ncl. ventrocaudalis anterior internus, V.im. Ncl.ventrointermedius, V.o.e. Ncl. ventro-oralis externus, V.o.i. Ncl.ventro-oralis internus

F

considerably complicates the interpretation of the molec-ular basis of this disease.

One possibility to explain neuronal degeneration incortical and subcortical structures in Huntington’s diseaseis to compare the numerical nerve cell loss with known fi-bre connections between affected structures. A strong nu-merical correlation between nerve cell loss of two inter-connected grisea provides arguments for anterograde, ret-rograde, or transneuronal mechanisms in neuronal degen-eration. The MD is reciprocally connected with the pre-frontal cortex. The MD in primates receives afferentsfrom, and emits topographically ordered efferents to, theprefrontal cortex [9, 35, 72, 95]. Methodological difficul-ties in case reports after traumatic, vascular, and tumour-

associated cortical or thalamic lesions [3, 11, 31, 39, 45,67, 71, 94, 104] have prevented a detailed description ofreciprocal thalamocortical or corticothalamic fibre con-nections in humans. However, the main topographic rela-tionships between MD subnuclei and cortical areas are ap-parently similar in man and monkey. Thalamocortical af-ferents terminate in layer IV and deep layer III of the pre-frontal cortex; prefrontal layer VI cells and, to a lesser ex-tent, layer V pyramidal cells emit corticothalamic effer-ents [35]. In a previous study, we have found a mean 33%overall cortical neurone loss in five HD patients (includedin this study) in combination with pallor of layers III andV and attenuation of layers VI [48]. These qualitative aswell as quantitative results point to possible neuronal loss

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Fig.5 Gallocyanin-stained neurones and glia from ventrolateralMD region in 58-year-old female control. Contrast of individualneurones is slightly less in 500 to 600 micrometer thick gallo-cyanin-stained sections than in 10 µm thick paraffin sections butstaining of neuronal nucleoli and nuclei of glial cells is sufficientfor quantitative analysis. In addition, the majority of the depictedcells are out of focus due to the unusual section thickness

Fig. 6 Gallocyanin-stained neurones and glia from correspondentregion as in Fig. 5; female 54-year-old HD patient

Fig. 7 Tissue from parallel formalin-stored frozen section afterparaffin embedding and staining with Gallyas’ [34] impregnationfor the demonstration of fibrous (reactive) astroglia. Section thick-ness 12.5 µm. Arrows point to three MD neurones. Female HD pa-tient, age 54 years, ventrolateral MD region corresponding to thatin Fig. 6

in the MD. Indeed, our quantitative investigations re-vealed a significant mean 23.8% nerve cells loss in theMD of HD patients (Table 1). The neurone loss in MDwas the lowest of all regions so far studied. In a recentstudy, van Eden et al. [105] found a 14% neurone loss inthe MD of rats after aspiration lesions of the prefrontalcortex in neonatal animals and a 21% neurone loss afterprefrontal cortical lesions in adult animals.

There is an obvious mismatch between total cor-tical neurone loss (–33%) and that of the MD nucleus (–23.8.%). The most likely explanation of the cortical ver-sus thalamic neuronal deficit is a preferential corticallayer III nerve cell loss. Layer III pyramidal cells areknown to project to neighbouring and far-distant corticalregions [8, 17, 82] as well as to the striatum [4, 36, 54, 55]and not to the MD. Nevertheless, global attenuation of in-fragranular layers resulting, nerve cell loss in cortical lay-ers V and VI [48, 96, 97] supply additional arguments foranterograde, retrograde or transneuronal nerve cell loss inthe MD of HD patients. Missing direct cortical input bydegenerating corticothalamic afferents could induce neu-ronal degeneration of MD neurones. Degeneration of cor-tical target neurones could be associated with death ofthalamic projection neurones. Further evidence can be de-rived from the presence of reactive astroglial cells in theMD of HD patients (Fig. 7). Nevertheless, the total glialcell number decreased in HD (see Table 1). This is similarto a decrease of total glial cell number in the striatum [48,58]. It is not known whether both glial types, astrocytesand oligodendrocytes, decrease equally in number orwhether this decrease is mainly due to oligodendroglial

degeneration. In contrast to the striatum, the glial indexremained nearly unchanged in MD. Therefore, neuronaland glial loss must balance each other in the course ofHD-associated MD atrophy. The positive staining resultswith Gallyas’ impregnation for fibrous astroglia in paraf-fin sections through the MD (Fig. 7) favour the view thatthe surviving astroglial cells become reactive to phagocy-tose and to form a glial scar that compensates tissue lossafter perikaryal, dendritic, and axonic degeneration. As-troglia is known to have a scavenger function and to removesynapses in diverse pathological conditions [18, 19, 74].

The absence of astrogliosis in the prefrontal cortex ofHD patients [96] and relative preservation of layer IV [48]perhaps indicate persistent connections of thalamocorticalneurones which project to deep layer III and layer IV [35].The 23.8% MD nerve cell loss would then be mainly a re-action to degenerating cortical projection neurones. Con-nections of MD neurones with subcortical structures, suchas the amygdala, could prevent progressive nerve cell lossin MD. The thalamic centromedian-parafascicular com-plex, on the other hand, receives cortical as well as striatalafferents. Since both structures are heavily affected inHD, nerve cell loss in the centromedian-parafascicularcomplex is more pronounced with 55% neuronal degener-ation [49].

Nerve cell loss in MD in diseases other than HD

Neurone loss in MD is not confined to HD. It has alsobeen described in schizophrenics by Pakkenberg [79].

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Table 1 Synopsis of quantitative data obtained for both HD patients and controls (Contr. controls, CV coefficient of variation, f females,HD Huntington’s disease, m males, SD standard deviation, Vol. MD (uncorrected) volume of MD). For interpretation see text

Diagnosis, Age Neurone- Glial Glial Vol. MD Neurone den- Glial densitysex (years) number number Index (mm3) sity (N/mm3) (N/mm3)

NT GT NV GV

HD f 36 1,995,858 13,286,548 6.66 365 5,467 36,395HD m 40 2,331,368 16,730,499 7.18 467 4,996 35,855HD m 41 2,151,983 14,390,462 6.69 417 5,167 34,551HD f 54 2,433,598 16,940,268 6.96 464 5,243 36,494HD f 62 2,657,913 13,002,912 4.89 536 4,958 24,256HD m 62 1,978,498 16,832,947 8.51 423 4,678 39,799HD m 72 2,378,026 16,048,631 6.75 489 4,863 32,819

Mean 52.4 2,275,321 15,318,895 6.81 451 5,053 34,310SD 13.7 247,162 1,722,524 1.06 55.5 261 4,916CV 0.109 0.112

Contr. m 36 3,01,5455 22,591,749 7.49 644 4,685 35,099Contr. f 46 3,011,916 25,090,625 8.33 612 4,918 40,972Contr. m 49 2,872,716 24,003,100 8.36 620 4,636 38,740Contr. f 53 2,849,611 18,801,515 6.60 531 5,365 35,395Contr. f 58 2,892,974 20,653,696 7.14 587 4,926 35,167Contr. m 58 3,352,495 24,169,173 7.21 705 5,708 41,152Contr. m 75 2,901,149 17,185,197 5.92 528 4,754 34,722

Mean 53.6 2,985,188 21,785,008 7.29 604 4,968 36,028SD 12.1 174,710 2,986,678 0.88 63 336 2,857CV 0.059 0.137

This author found a 40% reduction in total neurone num-ber, a 44% reduction in astrocyte number, and a 45% re-duction in oligodendrocyte number. Estimated neuronedensities in MD in control cases were similar in Pakken-berg’s estimates (5,311 neurones/mm3) [79] to those pre-sented here (4,968 neurones/mm3). However, total neu-rone numbers in MD in control cases were considerablylower in Pakkenberg’s estimates (1.81 × 106) [79] than inour estimates (2.985 × 106). In addition, estimated MDvolumes in control cases were on average 43% lower inPakkenberg’s estimates [79] compared with those pre-sented here. The most likely explanation is a different de-lineation of the MD by Pakkenberg in 5-mm-thick forma-lin-fixed slices prior to embedding and staining. Pak-kenberg did not observe a consistent elevation of as-troglial cell number in the MD of schizophrenics whichwould indicate neurodegenerative processes during thecourse of the illness. At variance with Pakkenberg [79], Bogerts [13] failed to demonstrate volume differ-ences in MD volume of controls and schizophrenics in theVogt collection. Xuereb et al. [116] found a nearly identi-cal volume of the MD in their five control cases (602 ±49.0 mm3) compared with our results (604 ± 63 mm3,Table 1). The total neurone number was considerablyhigher in the study of Xuereb et al. (3,916 ± 296 × 103 vs2,985 ± 174 × 103 neurones in the present study). SinceXuereb et al. [116] used a lower 250 × magnification (pos-sible confusion of nuclear heterochromatin with nucleoli)and the authors did not correct nucleolar counts, their datamight be biased.

Further, the thalamic MD is also affected in fatal fa-milial insomnia [16, 61, 62, 80, 87, 92] and in Wernicke-Korsakoff syndrome [22, 39, 68, 102, 106]. In the lattersyndrome we found a tremendous decrease of MD neu-rones from 2,539,406 neurones in the least affected caseto a minimum of 209,829 neurones in a 59-year-old fe-male [50]. To our surprise, we could not detect laminarnerve cell loss in the prefrontal cortex of Wernicke-Kor-sakoff patients. Our observations are in agreement withthose of Colmant [22] on a larger sample of Wernicke-Kor-sakoff patients. Traumatic, vascular and tumour-inducedlesions of MD have been reviewed by Markowitsch [63].Memory disturbances appear as the most likely common,although disputed, deficit of MD pathology in these unre-lated diseases.

Correlation of nerve cell loss in MD with clinical symptomatology

In contrast to our detailed knowledge of corticothalamicand thalamocortical projections, knowledge regarding thefunctional significance of first order and higher order thal-amic relay is sparse [93]. Considering the topography andfibre connections of MD subnuclei, multiple clinical de-ficits, including visceral-autonomic, visuo-spatial, mnestic,and social-emotional disturbances, appear likely after dif-fuse MD lesions [9, 117]. In at least half of HD patientsbehavioural and emotional abnormalities precede the ap-

pearance of neurological signs [29]. Interestingly, in threeof our seven patients, psychiatric diagnoses includingschizophrenia-like psychosis, behavioural abnormalities,and paranoia were given prior to the manifestation of in-voluntary choreic movements [48, 49].

Without detailed knowledge of neuronal loss in MDsubnuclei and their prefrontal cortical projection areas theassociation of neuronal deficits with behavioural or neu-rological deficits must remain incomplete. Furthermore,the morpho-functional correlation is complicated by nervecell loss in regions other than the cortex and MD. Thisnerve cell loss is most marked in the striatum [25, 48, 58,107], and the thalamic centromedian-parafascicular com-plex [49], it is also considerable in the globus pallidus[58] and slightly higher in the subthalamic nucleus [58]than in MD. In addition, the quantitative data do not pro-vide a reliable indication of the time course of nerve celldegeneration. Striatal neurone loss is the neuropathologi-cal hallmark of advanced stages of HD but neuroimagingstudies point to the early involvement of the frontal cortexin HD [64, 90] and reduced striatal metabolism is recog-nisable prior to striatal atrophy [37, 56, 118].

One of the earliest signs in HD, which was also used todiagnose presymptomatic HD carriers in the absence ofreliable genetic markers, is oculomotor disturbances, es-pecially a disturbed generation of saccades [7, 12, 14, 59,60, 76, 99, 103]. In primates several fronto-subcorticalpathways are engaged in oculomotor regulation. Thesesegregated and parallel pathways include the oculomotorcircuit [1,2] and pathways from area 8 to the thalamicparafascicular nucleus [53, 57, 98]; the parafascicular nu-cleus, in turn, receives additional afferents from the supe-rior colliculus [43, 81]. It emits efferents to both thefrontal eye field [53] and MD [89]. Neurone loss in majorrelay stations of these parallel and segregated circuits(striatum > 80%, centromedian-parafascicular complex > 50%, MD > 20%) in the course of HD could explain vi-suomotor disturbances which are frequently diagnosed inearly stages of HD and in at-risk relatives.

Conclusions

We consider the overall 33% cortical neurone loss, pallorand widening of lamina V, attenuation of lamina VI in theprefrontal cortex, 23.8 % neurone loss, and presence of fi-brous astroglial cells in MD as the morphological mani-festation of interrupted fronto-subcortico-frontal neuronalcircuits. We could not establish a linear correlation be-tween cortical/thalamic nerve cells loss and behaviouralor neurological deficits. Instead, we must learn to think inmultiple segregated and parallel neuronal circuits, similarto those in the cerebral cortex [69, 70] and consider con-founding effects such as plasticity [38, 97] or interindivid-ual variability in connectivity of the human brain. Theclassification of intellectual impairment in late stage ofHD as a “subcortical dementia” is no longer tenable [30,65, 115]. Recent excitotoxic animal models of HD em-phasised the major role of striatal neurodegeneration [10,

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23, 28] in the pathogenesis of HD. Future investigationsshould also focus on mechanisms and time course of neu-ronal degeneration in regions other than the striatum. Hy-potheses on the aetiology and pathogenesis of HD shouldinclude the cerebral cortex and subcortical grisea. Theo-retically, it is also possible that primary cortical (degener-ating layer III and layer V corticostriatal neurones) or sub-cortical (degenerating cells of centromedian-parafascicu-lar complex projecting to the striatum) pathology inducesecondary heavy neuronal degeneration in the striatum.

Acknowledgements The technical assistance of Mrs. E. Gößmannis gratefully acknowledged. This study was supported by a grantform the Deutsche Forschungsgemeinschaft (He 1430/4-1; BMH1-CT 94-1563 and by Synthelabo Arzneimittel GmbH, D-12067Berlin).

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