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Research Report

Distribution of the monocarboxylate transporter MCT2 inhuman cerebral cortex: An immunohistochemical study

Oriana Chirya,b, William N. Fishbeinc, Natalya Merezhinskayac, Stéphanie Clarked,e,Ralf Galuskea,b, Pierre J. Magistrettid,f,g, Luc Pellerind,⁎aMax Planck Institute for Brain Research, Deutschordenstraβe 46, 60528 Frankfurt am Main, GermanybTechnische Universiät Darmstadt, Institut für Zoologie, Schnittspahnstrasse 3, 64287 Darmstadt, GermanycBiochemical Pathology Division, Environmental and Toxicologic Pathology Department, Room M093C, Armed Forces, Institute of Pathology,Alaska Avenue and 14th Street NW, Washington, DC 20306-6000, USAdDépartement de Physiologie, Université de Lausanne, rue du Bugnon 7, 1005 Lausanne, SwitzerlandeService de Neuropsychologie et de Neuroréhabilitation, CHUV, Université de Lausanne, 1011 Lausanne, SwitzerlandfCenter for Psychiatric Neuroscience, CHUV, 1011 Lausanne, SwitzerlandgBrain Mind Institute, EPFL, 1015 Lausanne, Switzerland

A R T I C L E I N F O

⁎ Corresponding author. Fax: +41 21 692 5595.E-mail address: luc.pellerin@unil.ch (L. Pe

0006-8993/$ – see front matter © 2008 Elsevidoi:10.1016/j.brainres.2008.06.025

A B S T R A C T

Article history:Accepted 2 June 2008Available online 18 June 2008

The monocarboxylate transporter MCT2 belongs to a large family of membrane proteinsinvolved in the transport of lactate, pyruvate and ketone bodies. Although its expression inrodent brain has been well documented, the presence of MCT2 in the human brain has beenquestioned on the basis of low mRNA abundance. In this study, the distribution of themonocarboxylate transporter MCT2 has been investigated in the cortex of normal adulthuman brain using an immunohistochemical approach. Widespread neuropil staining in allcortical layers was observed by light microscopy. Such a distribution was very similar inthree different cortical areas investigated. At the cellular level, the expression of MCT2 couldbe observed in a large number of neurons, in fibers both in grey and white matter, as well asin some astrocytes, mostly localized in layer I and in the white matter. Double stainingexperiments combined with confocal microscopy confirmed the neuronal expression butalso suggested a preferential postsynaptic localization of synaptic MCT2 expression. A fewastrocytes in the grey matter appeared to exhibit MCT2 labelling but at low levels. Electronmicroscopy revealed strong MCT2 expression at asymmetric synapses in the postsynapticdensity and also within the spine head but not in the presynaptic terminal. These data notonly demonstrate neuronal MCT2 expression in human, but since a portion of it exhibits adistinct synaptic localization, it further supports a putative role for MCT2 in adjustment ofenergy supply to levels of activity.

© 2008 Elsevier B.V. All rights reserved.

Keywords:Energy metabolismLactatePostsynaptic densityNeuronAstrocyteAstrocyte–neuron lactate shuttle

llerin).

er B.V. All rights reserved.

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1. Introduction

Monocarboxylate transporters (MCTs) form a family of 14transmembrane proteins allowing specific metabolites, nota-bly lactate, pyruvate and the ketone bodies (acetoacetate andβ-hydroxybutyrate), to cross membranes (for review seeHalestrap and Meredith, 2004). Three isoforms, MCT1, MCT2and MCT4, have been described in the central nervous systemof rodents (Pierre and Pellerin, 2005). At the cellular level,MCT1 was found to be expressed by endothelial cells formingblood vessels, by glia limitans as well as by astrocytes(Gerhart et al., 1997; Hanu et al., 2000; Pierre et al., 2000).MCT4 is present exclusively in astrocytes (Rafiki et al., 2003;Pellerin et al., 2005). By contrast, MCT2 is the predominantneuronal monocarboxylate transporter (Bergersen et al., 2001;Debernardi et al., 2003; Pierre et al., 2000, 2002).

Little information are available so far about the expressionand distribution of monocarboxylate transporters in thehuman brain. One developmental study showed that bothMCT1 and MCT2 appear during early gestational stages ondifferent elements of the fetal brain including blood vessels,astrocytes and neurons (Fayol et al., 2004). In the adult humanbrain, two immunohistochemical studies showed that MCT1is expressed on blood vessels as well as by astrocytes indifferent cortical areas (Chiry et al., 2006; Froberg et al., 2001).No information is available yet about MCT4 protein expres-sion. Concerning MCT2, no immunohistochemical study hasbeen performed so far. Although, a northern blot analysis hadsuggested that in contrast to the rodent brain, MCT2might notbe expressed in human tissues including the brain (Price et al.1998), western blotting with specific anti-MCT2 antibodydemonstrated quite significant levels of that transporter inhuman brain cortex membranes, along with those of severalother tissues (Fishbein et al, 2002). However, that paperincluded an immunohistochemical study of skeletal muscleonly. Thus for the moment, the identity of the monocarbox-ylate transporter expressed by neurons in the human brain, ifany, remains uncertain.

Independently, several observations suggest that adulthuman brain cells, and particularly neurons, would requirethe presence of monocarboxylate transporters. Indeed, lactateis present within the human brain parenchyma at a concen-tration estimated around 5 mM with the microdialysis zeroflow technique (Abi-Saab et al., 2002). Moreover, changes inlactate concentrations upon activation have been measuredand suggest that lactate could be produced and/or consumedby human brain cells (Mangia et al., 2003; Prichard et al., 1991).Identification of a monocarboxylate transporter expressed byneurons in the human brain would provide further confirma-tion that the recently developed view of neuroenergeticsinvolving intercellular lactate fluxes, elaborated from work onrodents, can be extended to humans.

2. Results

The distribution of the monocarboxylate transporter MCT2 inthe cerebral cortex was investigated using post-mortem tissuefrom eight normal human individuals. Primary and secondary

cortical areas from the auditory cortex but also from the visualcortex were analyzed. Brain areas and cortical layers wereidentified using Nissl staining processed on nearby sections.The immunolabelling intensity and distribution observed wassimilar among all cases studied.

Light microscopy analysis of MCT2-stained sectionsshowed a light and diffuse neuropil expression across allcortical layers (Fig. 1). Staining of the white matter underlyingthe cortex was less intense than the grey matter labelling.Comparison between cortical areas did not reveal strikingdifferences in staining intensity. This was true between areasbelonging to different hierarchical levels, i.e. primary auditorycortex vs. higher order auditory cortex (Fig. 1), but also forareas belonging to the same hierarchical level, i.e. primaryauditory vs. primary visual cortex (not shown). Besidesneuropil staining, MCT2-positive fibers were visible in thewhite matter and in the cortex. Cortical fibers were morenumerous in the granular and infragranular layers in all areasinvestigated (Fig. 1). Positive fibers in the cortex were mostlyoriented perpendicularly to the pial surface (Fig. 1A), except forsome fibers running both horizontally and radially in layer IV(Fig. 2C).

At higher magnification, a few more MCT2-stainedelements could be identified. In general, cell bodies ofneurons were lightly stained as compared to fibers (Fig. 2A).Few labelled axons or dendrites protruding from these cellbodies were usually observed. However, in specific corticallayers, several cell bodies of pyramidal and non-pyramidalneurons were darkly stained and positive processes wereobserved (Fig. 2B at high magnification). Often, the expres-sion of MCT2 appeared as puncta in the cytoplasm ofneuronal-like elements (i.e. apical dendrites of positive pyra-mids, Fig. 2B). Numerous fibers were observed in layer IVexhibiting a perpendicular or horizontal orientation com-pared to the pial surface (Fig. 2C) or in the white matterunderlying area AI (Fig. 2D) and V1 (not shown). In addition toneurons and neuronal elements, several fibrous astrocytes inlayer I (Fig. 2E) and in the white matter (Fig. 2F at highmagnification) could be identified. No staining was observedwhen the MCT2 antibody was omitted (Fig. 2G).

Neuronal expression of MCT2 (Figs. 3A–D) was confirmedby performing double labelling with neuronal markers. First,when performed in combination with an antibody againstNeuN, a protein specifically expressed in neuronal nuclei,MCT2 staining was clearly present in many cell bodiessurrounding NeuN-positive nuclei (Figs. 3A1–3). In the caseof fibers that were previously shown to express MCT2, it couldbe demonstrated that they are neuronal processes as perfectcolocalization was observed between MCT2 and the neuronalcytoskeletal marker MAP2 (Figs. 3B1–3). In addition to cellbodies and fibers, a strong punctiform labelling in the neuropilarea was also observed (Fig. 3A1). In order to determinewhether it belongs to specific synaptic elements, doublelabellings with synaptic markers were performed. No coloca-lization could be found between MCT2 and the presynapticmarker synaptophysin (Figs. 3C1–3). By contrast, an occasionalcolocalization was observed between MCT2 and the postsy-naptic marker PSD95 (Figs. 3D1–3). As opposed to layer I and inwhite matter, few astrocytes stained for MCT2 in other layersof the grey matter. Double labellings with the astrocytic

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marker GFAP confirmed that only rare astrocytes expressedMCT2 in this area (uncommon double labelled astrocytesshown on Fig. 3E1–3). Those few astrocytes containing lowlevels of MCT2 were often found in close contact with MCT2-positive neuronal cell bodies.

To investigate the subcellular localization of MCT2 inneuronal elements, we performed an electron microscopicanalysis on ultrathin stained sections. In recognizable asym-metric synapses, MCT2 labelling was preferentially located inthe postsynaptic element, associated with the postsynapticdensity aswell as distributedwithin the spinehead (Figs. 4A–B).By contrast, in the negative control, the density of silverparticles was low and homogenously distributed when theprimary antibody was omitted (Fig. 4C). In certain cases, a fiberloadedwithMCT2-associatedsilverparticles couldbe identified(Fig. 4D).

3. Discussion

Several studies in rodents have documented the strongexpression of the monocarboxylate transporter MCT2throughout the central nervous system both at the mRNAand protein levels (Bergersen et al., 2001, 2002; Koehler-Stecet al., 1998; Pellerin et al., 1998; Pierre et al., 2000, 2002). Fewstudies on MCT expression have been performed in thehuman brain so far, but based on the low mRNA levelsdetected, it was postulated that MCT2 protein expressionmust be very low, if present at all (Price et al. 1998). Datareported here demonstrate for the first time that MCT2protein expression is strong and widespread in differentcortical areas of the human brain. At the cellular level, it isclear that MCT2 is a prominent neuronal transporteralthough it could be detected also in some astrocytes,particularly in the white matter. Strong expression in fibersor axons, particularly in certain areas, is also a commoncharacteristic of MCT2 expression in the rodent brain (Pierreet al., 2002; Tekkök et al., 2005). At the subcellular level,colocalization with the postsynaptic protein PSD95 but notwith the presynaptic protein synaptophysin similarly pro-vided an indication that MCT2 is enriched postsynaptically inthe human brain (Pierre et al., 2002). Just like in rodents(Bergersen et al., 2001, 2002, 2005), this specific localization atasymmetric synapses could be confirmed by electron micro-scopy. Based on all these observations, it appears that MCT2expression and distribution is remarkably similar betweendistinct species. Thus, it further strengthens the notion thatMCT2 is the predominant neuronal monocarboxylatetransporter.

The confirmation that MCT2 represents the main neuro-nal monocarboxylate transporter in the human brain com-plements previous descriptions of MCT1 being a major

Fig. 1 –Laminar distribution of MCT2 immunolabelling in thehuman primary auditory area (A) and in the higher orderauditory area STA (B; Rivier and Clarke, 1997) revealed byimmunoperoxidase staining viewed with light microscopy.Bars on the left side indicate transitions between I/II, III/IV,IV/V and VI/white matter. Scale bar=500 μm.

Fig. 2 –Cellular distribution ofMCT2 in humanprimary auditory cortex as revealed by immunoperoxidase staining viewedwithlight microscopy. Lightly stained MCT2-positive neurons and strongly labelled fibers in layer IV (A). MCT2-positive pyramidalneuron in layer III at highermagnification (B). Note that theMCT2 labelling has a punctiformappearance, especially in the apicaldendrite. MCT2-positive fibers with a tangential and/or perpendicular orientation in layer IV (C) and in the white matterunderlying the area AI (D). MCT2-positive astrocytes in layer I (E) and a MCT2-positive fibrous astrocyte in the white matter athigher magnification (F). Negative control (G). Scale bar=50 μm.

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monocarboxylate transporter found both on human micro-vessels and astrocytes (Chiry et al., 2006; Froberg et al., 2001).The distribution of these two monocarboxylate transporterisoforms in the human brain is consistent with a number of

observations related to the importance of lactate as asupplemental energy substrate for the human brain underspecific circumstances (Dalsgaard, 2006; Pellerin et al., 2007).Thus, it was shown that when plasma lactate is raised to

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levels reached during moderate exercise (Smith et al., 2003) orrises as a normal consequence of exercise (Dalsgaard et al.,2004), it is used by brain cells. The presence of MCT1 onendothelial cells forming blood vessels and MCT2 on neuronsprovides the necessary conditions to facilitate lactate entryinto the brain and uptake by active neurons under suchconditions. Brain activation per se was shown to lead tobiphasic changes in lactate levels within the brain parench-yma (Prichard et al., 1991; Mangia et al., 2003). Recenttheoretical analysis has demonstrated that such changes inintracerebral lactate levels upon activation can be explainedby production from astrocytes and uptake by neurons (Aubertet al., 2005, 2007). Again, the described distribution of MCT1 inastrocytes and MCT2 in neurons would be consistent with anet transfer of lactate between the two cell types in the humanbrain as well. In addition, the lower Km displayed by MCT2compared to MCT1 is consistent with a net flux of lactate fromastrocytes to neurons (Bröer et al., 1997, 1999).

Despite an apparently low mRNA abundance, the MCT2protein is highly expressed, mainly by neurons, and widelydistributed throughout the human brain. Such a discrepancybetween mRNA and protein expression suggests a particularregulation of translation (Zhang et al., 2007). Indeed, it hasalready been suggested that MCT2 has a putatively complexexpression regulation with multiple transcripts found both inrodent and human brain (Jackson et al., 1997; Lin et al., 1998;Pellerin et al. 1998) most likely arising from alternativepromoter usage as well as from alternative splicing (Zhang etal., 2007). Moreover, a specific translational control of MCT2expression by noradrenaline, insulin and IGF-1 involving theactivation of the PI3K/Akt/mTOR/S6K pathway has beendemonstrated in mouse cortical neurons in vitro (Chenal andPellerin, 2007; Chenal et al., 2008; Pierre et al., 2003). Consider-ing the data presented here, it is likely that the human brainwill exhibit a similarly complex regulation. Such featuresmight subserve a critical role for MCT2 in adapting energysupply to demand not only upon acute activation but also forprolonged changes in activity associated with brain plasticity.

4. Experimental procedures

4.1. Preparation of tissues

Tissue from both hemispheres of five normal human brains,obtained through a donor program at the Institute of CellBiology andMorphology of the Faculty of Biology andMedicineat the University of Lausanne, was investigated in the presentstudy. Subjects were respectively 81, 90, 83, 87 and 83 yearsold. Two were males and three females. The post-mortemdelay was between 6 and 12 h. These brains were included in aprevious study investigating the distribution of MCT1 (Chiry etal., 2006). Brain sections from each case were processed forlight and confocal microscopy. Tissue from 4 additionalhemispheres from three different subjects was obtainedfrom the Pathology Department of the Goethe University ofFrankfurt in Frankfurt am Main. The three cases wererespectively 74, 59, and 78 years old. One was female andtwo were males. Brain sections from these three new caseshave been investigated with lightmicroscopy and one of them

also with electronmicroscopy. None of the subject included inthis study was known to have either neurological or psychia-tric diseases and all died from causes unrelated to the nervoussystem (cardiac failure). Macro- and microscopical examina-tion of all brains did not reveal any lesions.

The fixation and preparation procedure has been pre-viously described in detail (Chiry et al., 2006). Three of thebrains were perfused with 4% paraformaldehyde in 0.1 Mphosphate buffer (pH 7.4) through the basilary artery and thetwo internal carotid arteries for a duration of 60 min afterremoving the brain from the skull. The post-fixation wasperformed by immersion in 4% paraformaldehyde in 0.1 Mphosphate buffer (pH 7.4) for 10 h. The other brains wereremoved from the skull, rinsed in 0.9% NaCl solution and fixedby immersion in 4% paraformaldehyde in 0.1 M phosphatebuffer (pH 7.4) for 24 h. Blocks including the supratemporalregion, except for the block processed for electronmicroscopy,were then cryoprotected with graded sucrose concentrations(10–30%) in phosphate buffer at 4 °C until they sank.

Brains were cut using a cryostat in coronal sections of40 μm or 60 μm. They were conserved in ethylene glycol at−20 °C until utilization. The block processed for electronmicroscopy was cut using a vibratome in coronal sections of30 μmand immediately processed for immunohistochemistry.

4.2. Immunohistochemistry

Free-floating sections from the supratemporal region, thesuperior temporal gyrus and the occipital lobe were processedfor MCT2 immunohistochemistry using polyclonal antibodies.The antibody was raised against the last 16 C-terminal aminoacids of the human MCT2 transporter (Fishbein et al., 2002).The synthetic peptide used to generate the antibody wascomposed of a N-terminal cysteine followed by amino acids463–478 of the human MCT2 transporter with the followingsequence: C-KVSNAQSVTSERETNI. Sections were washed inTris-buffered saline (TBS 0.05M, pH 7.4) and then treated with1% hydrogen peroxide in methanol for 10 min. Sections werethen washed twice for 10 min in TBS and permeabilized for10 min in TBS containing 1% Triton X-100. After washing,sections were preincubated 1 h in TBS containing 0.5% caseinand 0.05% sodium azide to prevent non-specific staining.Sections were then incubated at 4 °C in 0.25% bovine serumalbumin (BSA) in TBS containing the antibody against MCT2(dilution 1:200, corresponding to 12 mg/ml). Optimal dilutionsand incubation times were determined in preliminary tests.Optimal incubation time was 20 h. Sections were then washed3 times in TBS and incubated 2 h in the secondary antibodyanti-rabbit IgG (Jackson; West Grove, U.S.A.) diluted to 1:200 in0.25% BSA in TBS, followed by 2 h in rabbit peroxidaseantiperoxidase (Jackson, West Grove, U.S.A.) diluted to 1:500in 0.25%BSA inTBS. Boundantibodywasdetectedwith glucoseoxidase-nickel solution containing 3,3′diaminobenzidine tet-rahydrochloride as chromogen.

In control experiments, when the primary antibody wasomitted, no staining was detected. In a second controlexperiment for MCT2, the primary antibody was incubatedwith an excess of the corresponding control peptide (obtainedfrom AlphaDiagnostic International; MCT2 control peptide),and no staining was detected. Sections near (within 400 μm)

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Fig. 4 –Subcellular localization of MCT2 in neurons of the human cerebral cortex revealed by electronmicroscopy.(A,B) Immunolabellings forMCT2 at asymmetric synapses. Silver grains representingMCT2 proteins are located preferentially on thepostsynaptic side, associated with the postsynaptic density or within the spine head. (C) Control with omission of the primaryantibody. In such case, background levels were low and no specific enrichment in any compartment was observed.(D) Immunolabelling forMCT2showingstrongexpression inaneuronalprocess.Arrowhead,postsynapticdensity. Scalebar=0.5 µm.

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those processed for metabolic markers were stained withcresyl violet.

4.3. Double immunostaining

To determine the cellular and subcellular localization ofMCT2,adjacent or nearby sectionswere double stained for bothMCT2

Fig. 3 –Cellular and subcellular localization of MCT2 (green; A1–Efluorescence immunolabelling viewed with confocal microscopy.(red; A2) and double labellingwithMCT2 (A3). Immunolabelling olabelling with MCT2 revealing colocalization (yellow; B3). Immunsynaptophysin (red; C2) and double labelling with MCT2 (C3). Immdouble labelling with MCT2 revealing partial colocalization (yello(red; E2) double immunolabelling with MCT2 revealing very restroptical single sectionwhile the other pictures represent the superplane. Scale bar=50 μm.

and specific marker proteins. Free-floating sections processedfor immunohistochemistry of MCT2 (diluted 1:200) were thenstainedwithmonoclonal antibodies against NeuN, amarker ofneurons (1:800 dilution, Sigma), against the glial fibrillary acidprotein (GFAP, dilution 1:800, monoclonal, Sigma), againstmicrotubule associated protein 2, a neuronal cytoskeletalprotein (MAP2, dilution 1:500, polyclonal, Abcam, Cambridge,

1) in the human primary auditory cortex as revealed byImmunolabelling with the neuronal nuclei marker NeuNf the neuronal cytoskeleton proteinMAP2 (red; B2) and doubleolabelling with the presynaptic terminal markerunolabelling of the postsynaptic protein PSD95 (red; D2) and

w; D3). Immunolabelling of the astrocytic marker GFAPicted colocalization (yellow; E3). Pictures C1–3 represent anposition of optical sections spaced 1μmprojected on a single

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UK), against postsynaptic density 95, a postsynaptic marker(PSD95, dilution 1:100, polyclonal, Abcam, Cambridge, UK), oragainst synaptophysin (1:100 dilution, monoclonal, Chemi-con). After washing in Tris-buffered saline (TBS 0.05M, pH 7.4),sections were preincubated for 15 min in TBS containingNormal Goat Serum and Normal Donkey Serum (both diluted1:10) and 0.1% Triton X-100. Sections were then incubated at4 °C for 20 h inTBS containing both anti-MCT2 and anti-markerprotein antibodies. After washing in TBS, the MCT2 antibodywas revealed by a 1 h incubation with secondary biotinylatedgoat anti-rabbit IgG (Jackson; West Grove, U.S.A.) diluted to1:100 in TBS followed by 1 h in FITC conjugated avidin (VectorLaboratories) diluted to 1:100 in TBS. The same sections wereincubated 1 hwith cyanine cy3 conjugated donkey anti-mouse(Jackson, West Grove, U.S.A.) diluted 1:100 in TBS. To avoidautofluorescence, we counterstained sections with SudanBlack (Fluka, 1% in 70% ethanol). Sections were then mountedin Fluorsave reagent (Calbiochem) and photomicrographswere taken with a confocal laser scanning microscope (LeicaTCS NT). Images of 1024×1024 pixels were takenwith 25×, 40×,63× or 100× objectives. For each photomicrograph, stacks ofoptical sections spaced 1 μmwere collected and projected on asingle plane. Presence of colocalization was always verified onindividual 1 μm optical sections.

4.4. Electron microscopy

For electron microscopy analysis, sections were washed inTris-buffered saline (TBS 0.05 M, pH 7.4) and treated with 1%hydrogen peroxide in TBS for 10 min. They were then washedtwice for 10 min in TBS and rapidly frozen on a cold metalblock. Sections were preincubated 1 h in TBS containing 0.5%casein and 0.05% sodium azide to prevent non-specificstaining. They were then incubated at 4 °C in 0.25% bovineserum albumin (BSA) in TBS containing the antibody againstMCT2 (dilution 1:200, corresponding to 12 mg/ml) for 20 h.Sections were washed 3 times in TBS and incubated for 2 h inthe secondary antibody anti-rabbit IgG (Jackson; West Grove,U.S.A.) diluted to 1:200 in 0.25% BSA in TBS, followed by 2 h inrabbit peroxidase antiperoxidase (Jackson, West Grove, U.S.A.)diluted to 1:500 in 0.25% BSA in TBS. Bound antibody wasdetected with 3,3′diaminobenzidine tetrahydrochloride aschromogen. After staining, sections were post-fixed for 1 hin 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), andthen washed in 0.1 M phosphate buffer (pH 7.4) followed by asilver intensification and by a 20 min treatment with osmium(0.5% osmium in phosphate buffer). After dehydration, sec-tions were incubated in propylene oxide for 5 min, andembedded in Epon. After polymerization, 90 nm ultrathinsections were cut with a diamond knife. The ultrathinsections were then incubated in lead citrate and uranylacetateto enhance contrast. Sections were examined using a ZeissEM10 electron microscope (Oberkochen, Germany).

Acknowledgments

We thank Profs. E.Welker and J.P. Hornungwho gave us accessto the material obtained as part of a donor program at theInstitute of Cellular Biology and Morphology, University of

Lausanne. We thank Dr S. Momma who gave us access to thematerial obtained from the Pathology Institute of the GoetheUniversity in Frankfurt. We thank also W. Hofer for theelectron microscopy support. We express our gratitude to Dr.K. Pierre for reading the manuscript and for her experimentalsuggestions. This work was supported by the National SwissScience Foundation grant nos 3100-103895 (to SC) and 3100A0-112119 (to LP) as well as by a postdoctoral fellowship to OC.

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