distribution of a specific calcium-binding protein of the s100 protein family, s100a6 (calcyclin),...

Distribution of a SpecificCalcium-Binding Protein of the S100Protein Family, S100A6 (Calcyclin),

in Subpopulations of Neurons and GlialCells of the Adult Rat Nervous System

NORIFUMI YAMASHITA,1* EVELYN C. ILG,2 BEAT W. SCHAFER,2

CLAUS W. HEIZMANN,2 AND TOSHIO KOSAKA1

1Department of Anatomy and Neurobiology, Faculty of Medicine, Kyushu University,Higashi-ku, Fukuoka 812–8582, Japan

2 Department of Pediatrics, Division of Clinical Chemistry and Biochemistry,University of Zurich, CH-8032 Zurich, Switzerland

ABSTRACTS100A6 (calcyclin) is a member of the large S100 Ca21-binding protein family, considered

to activate several processes along the calcium signal transduction pathway including theregulation of cell growth, proliferation, secretion, and exocytosis. In the present study, thedistribution of S100A6 in the rat nervous system was examined by immunohistochemistrywith a goat antiserum against recombinant human S100A6, which recognizes the rat S100A6homologue. The main S100A6-immunoreactive elements were 1) neuronal somata anddendrites in some specific regions of the limbic system (e.g., the basolateral amygdaloidnucleus, ventral tip of the CA1-subicular border region, entorhinal cortex, and parasubicu-lum), most of which were identified as a subpopulation of pyramidal cells; 2) olfactory receptorcells and olfactory nerve fibers and terminals in the olfactory bulb; 3) some tracts of thehindbrain and spinal cord (e.g., the spinal trigeminal tract, solitary tract, dorsal root fibers,and the tract of Lissauer) and their terminals (e.g., the principal sensory trigeminal nucleus,spinal trigeminal nucleus, nucleus of the solitary tract, marginal zone, substantia gelatinosa,and proper sensory nucleus of the dorsal horn), as well as some sensory neurons of theirorigins in the dorsal root and trigeminal ganglia; 4) a subpopulation of astrocytes in the whitematter (e.g., the corpus callosum, cingulum, external capsule, internal capsule, and fimbria ofthe hippocampus) and around the ventricles; 5) some ependymal cells, especially around thecentral canal; and 6) Schwann cells. These results will improve our understanding of thediverse function of Ca21-binding proteins in the CNS. J. Comp. Neurol. 404:235–257,1999. r 1999 Wiley-Liss, Inc.

Indexing terms: limbic system; olfactory nerve; trigeminal ganglia; dorsal root ganglia;

immunohistochemistry

The family of S100 proteins is one of the largest subfami-lies of EF-hand calcium-binding proteins. To date, some 18different proteins have been assigned to the S100 proteinfamily (Schafer and Heizmann, 1996; Ilg et al., 1996b;Wicki et al., 1996). The S100 proteins have low molecularweights (10–12 kDa), have two EF-hand domains, andform dimers; most of them bind two calcium ions permonomer. The physical properties of S100 proteins (Pedroc-chi et al., 1994) suggest that they activate target proteinsand therefore influence cellular response along the calcium-signal-transduction pathway (Schafer and Heizmann,

Grant sponsor: Japanese Ministry of Education, Science and Culture:Grants-in-aid for General Scientific Research; Grant number: 09480213;Grant sponsor: Uehara Memorial Foundation; Grant sponsor: MitsubishiFoundation; Grant sponsor: Wilhelm Sander-Stiftung (FRG); Grant spon-sor: Biomed 2, Switzerland; Grant number: BBW 95.0215–1.

The term S100A6 is according to the new nomenclature of S100 proteinsproposed by Schafer et al. (1995).

*Correspondence to: Norifumi Yamashita, Department of Anatomy andNeurobiology, Faculty of Medicine, Kyushu University, Higashi-ku, Fukuoka812–8582, Japan. E-mail: ymst@a3rd. med. kyushu-u.ac.jp

Received 21 October 1997; Revised 31 August 1998; Accepted 9 Septem-ber 1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 404:235–257 (1999)

r 1999 WILEY-LISS, INC.

1996). Recently, S100A6, a member of the S100 Ca21-binding protein family, has been further characterized. Byusing nuclear magnetic resonance spectroscopy, the three-dimensional structure of S100A6 has been reported (Pottset al., 1995; Sastry et al., 1998), revealing a homodimericprotein fold that is novel among the EF-hand calcium-binding proteins. It is suggested that the dimeric form ofS100A6 is the active protein in vivo. The S100A6 gene is onhuman chromosome 1q21 (Schafer et al., 1995) clusteredtogether with most of the other S100 genes in a regionfrequently displaying alterations in transformed cells.S100A6 binds not only Ca21 but also Zn21 (Filipek et al.,

1990; Fohr et al., 1995), indicating that S100A6 might beimportant to the role of Zn21 in the central nervous system(CNS). Furthermore, annexins (Zeng et al., 1993), tropo-myosin (Golitsina et al., 1996), and p30 (Filipek andWojda, 1996) have been reported to be target proteins ofS100A6, through which S100A6 is considered to mediatediverse functions such as regulation of cell growth, prolif-eration, secretion, and exocytosis (Schafer and Heizmann,1996).

The human tissues containing high levels of S100A6mRNA are heart muscle, kidney, lung, thymus, and brain(Engelkamp et al., 1993). The level of S100A6 mRNA in the

Abbreviations

3V third ventricle4V fourth ventricle7n facial nerve or its root9n grossopharyngeal nerve10 dorsal motor nucleus of vagus10n vagus nerveab angular bundleac anterior commissureaca anterior commissure, anteriorACB accumbens nucleusaci anterior commissure, intrabulbaracp anterior commissure, posteriorAH anterior hypothalamic areaAHi amygdalohippocampal areaalv alveus of the hippocampusAmb ambiguus nucleusAD anterodorsal thalamic nucleusAOB accessory olfactory bulbAOD anterior olfactory nucleus, dorsal partAOP anterior olfactory nucleus, posterior partAOV anterior olfactory nucleus, ventral partAP area postremaApir amygdalopiriform transition areaAq aqueductBL basolateral amygdaloid nucleusbp brachium pontisBST bed nucleus of the stria terminalisCA1 field CA1 of hippocampusCA2 field CA2 of hippocampusCA3 field CA3 of hippocampuscc corpus callosumCC central canalCe central amygdaloid nucleuscg cingulumCG central graycp cerebral peduncle, basal partCPu caudate putamencu cuneate fasciculusCu cuneate nucleusD3V dorsal third ventricledf dorsal fornixdl dorsolateral fasciculus (tract of Lissauer)DG dentate gyrusDH dorsal horn of the spinal cordDLG dorsal lateral geniculate nucleusDM dorsomedial hypothalamic nucleusDMSp5 dorsomedial spinal trigeminal nucleusE/OV ependyma and subependymal layer around the olfactory

ventricleec external capsuleECIC external cortex of the inferior colliculusECu external cuneate nucleusEnt entorhinal cortexEPl external plexiform layer of the olfactory bulbf fornixfi fimbria of the hippocampusfmi forceps minor of the corpus callosumfmj forceps major of the corpus callosumg7 genu of the facial nerveGl glomerular layer of the olfactory bulbGL granular layer of the dentate gyrus

GP globus pallidusgr gracile fasciculusGr gracile nucleusGrO granular cell layer of the main olfactory bulbHiF hippocampal fissureic internal capsuleicp inferior cerebellar peduncleIPl internal plexiform layer of the olfactory bulbIR area infraradiataLD laterodorsal thalamic nucleuslEnt lateral entorhinal cortexLH lateral hypothalamic arealo lateral olfactory tractLS lateral septal nucleusLV lateral ventriclemcp middle cerebellar peduncleMD mediodorsal thalamic nucleusmEnt medial entorhinal cortexMG medial geniculate nucleusMi mitral cell layer of the main olfactory bulbml medial lemniscusML molecular layer of the dentate gyrusmlf medial longitudinal fasciculusMM medial mammillary nucleus, medial partMOB main olfactory bulbMVe medial vestibular nucleusON olfactory nerve layeropt optic tractox optic chiasmPa5 paratrigeminal nucleusPaS parasubiculumPaV paraventricular hypothalamic nucleus, ventral partpc posterior commissurePCRt parvicellular reticular nucleuspf posterior funiculusPir piriform cortexPn pontine nucleusPr5DM principal sensory trigeminal nucleus, dorsomedial partPrS presubiculumPRh perirhinal cortexPy pyramidal tractrf rhinal fissureS subiculums5 sensory root of the trigeminal nerveSC superior colliculusscp superior cerebellar pedunclesm stria medullaris of the thalamusSN substantia nigrasol solitary tractSol nucleus of the solitary tractsp5 spinal trigeminal tractSp5C spinal trigeminal nucleus, caudal partSp5I spinal trigeminal nucleus, interpolar partst stria terminalisTr triangular transition areaTu olfactory tubercleVH ventral horn of the spinal cordvhc ventral hippocampal commissureVLG ventral lateral geniculate nucleusVP ventral pallidum

236 N. YAMASHITA ET AL.

brain is lower than in the kidney, but higher than in theliver (Guo et al., 1990). The S100A6 protein level in thebrain estimated by enzyme-linked immunosorbent assay(ELISA) is about 280 ng/mg of soluble proteins (Zeng andGabius, 1991). In vitro analyses of nerve tissue-related celllines also suggest a possible importance of S100A6 in thenervous system. e.g., PC12 cells activated by nerve growthfactor (Leonard et al., 1987; Thompson and Ziff, 1989) andSchwann-like cells of retinoic acid-induced neuroblastoma(Tonini et al., 1991). However, S100A6 expression in thenervous system has not yet been analyzed systematicallyin detail, except in a brief report by Filipek et al. (1993) inthe rat CNS. Recently, we raised polyclonal antibodyagainst recombinant human S100A6 (Ilg et al., 1996a) andstarted a systematic survey of the distribution of thiscalcium-binding protein, with the intention of gaininginsight into the S100A6 functions in the CNS. The aims ofthis study were to localize S100A6 by immunohistochemis-try and to establish a detailed topographical map of theS100A6-immunoreactive (S100A6-IR) elements in the ratnervous system.

MATERIALS AND METHODS

Twenty-three adult male Wistar rats (specific pathogenfree, 110–300 g, 5–9 weeks old; Kyudo, Kumamoto, Japan)were used in the present study. All animals were treated inaccordance with the institutional guidance for animalwelfare. The animals were deeply anesthetized with pento-barbital sodium (50 mg/kg body weight) and perfusedtranscardially with phosphate-buffered saline solution(PBS) (pH 7.4), followed by a mixture of 4% paraformalde-hyde, 0.1% glutaraldehyde, and 0.2% picric acid in 0.1 MMillonig’s phosphate buffer (pH 7.4) (19 rats) or by 4%paraformaldehyde in the same buffer (4 rats). Brains,spinal cords, trigeminal ganglia, dorsal root ganglia, olfac-tory mucosa, and retina were left in situ for 1–2 hours atroom temperature, then removed from the skull, spinalvertebrae, and surrounding tissues, and finally cut into50-µm-thick serial sections on a vibratome (TechnicalProducts, St. Louis, MO). Whole brains were cut eithercoronally, horizontally, or parasagittally. Spinal cords werecut transversely. Trigeminal ganglia, spinal ganglia, olfac-tory mucosa, and retina were embedded in agar and thencut transversely. Sections were then rinsed several timeswith PBS. They were divided into sets of six consecutivesections. The first section of each set was processed forNissl staining, the second for immunohistochemistry forS100A6, and the third for counter Nissl staining combinedwith immunohistochemistry for S100A6. The remainingsections were used for immunohistochemistry for S100B,glial fibrillary acidic protein (GFAP), parvalbumin, and/orcalbindin D-28K (calbindin). Some selected sections wereexamined and drawn with a light microscope equippedwith a camera lucida apparatus (Nikon Optiphot [Nikon,Tokyo, Japan]) and photomicrographed with a light micro-scope (Zeiss Axiophot [Zeiss, Oberkochen, Germany]). Inaddition to these sets of differently processed sections,some sections from other series were processed by multiplefluorescent immunohistochemistry and examined with aconfocal laser scanning light microscope (CLSM; Bio-RadMRC 1000 [Bio-Rad, Richmond, CA] mounted on a Nikonlight microscope Optiphot) by using laser beams of 488,568, and 647 nm for excitation with appropriate filter sets(T1 and T2A dichroic filter block sets).

Negatives of light micrographs and camera lucida draw-ings were scanned by a 35 mm film scanner (Coolscan II,Nikon) and a flat-bed scanner (JX-330M, Sharp, Osaka,Japan), respectively, and images were transferred to apersonal computer (Power Macintosh 7500/100, Apple,Cupertino, CA). CLSM projection images were transferredto a personal computer (Power Macintosh 7500/100) byusing National Institutes of Health image software. Finalimages of camera lucida drawings were created by agraphic software (Canvas 3.5, Deneba Software, Miami,FL) using the scanned drawings as the templates. Forpreparing illustrations for presentation, selective imageswere processed by a image editing software (Adobe Photo-shop 3.0.5J, Adobe Systems, Mountain View, CA) andcombined into plates. Only the contrast and brightnesswere adjusted, and deliberate image processes were notdone. Figures were directly printed on a Pictrography(Fuji-Xerox, Tokyo, Japan). For identifying structures weconsulted the atlas of Paxinos and Watson (1997).

Nissl staining

Sections were collected on gelatin-coated glass slides,air-dried, and stained with toluidine blue or cresyl violet.After dehydration in graded series of ethanol, sectionswere cleared with xylene and mounted in M·X mountingmedium (Matsunami, Osaka, Japan).

Immunohistochemistry

Sections were rinsed with PBS and preincubated for30–60 minutes in 80% methanol containing 3% H2O2 toreduce endogenous peroxidase activity. They were washedin PBS three times for 15–20 minutes, incubated for 1 hourin 1% bovine serum albumin (BSA) in PBS containing0.3% Triton X-100, and then incubated for 48–96 hourswith primary antibodies in 1% BSA in PBS containing0.3% Triton X-100 and 0.05% sodium azide at roomtemperature. The primary antibodies were 1) goat poly-clonal anti-recombinant human S100A6 (1:10,000–20,000;Ilg et al., 1996a); 2) rabbit polyclonal anti-bovine S100(mostly S100B) (1:5,000; Dakopatts, Copenhagen, Den-mark); 3) mouse monoclonal anti-GFAP (1:5,000; Boeh-ringer, Mannheim, Germany); 4) rabbit polyclonal anti-rat

Fig. 1. Specificity of polyclonal antiserum against S100A6 wastested by Western blot analysis. A: SDS-PAGE of human recombinantproteins (lane a) S100A1, (land b) S100B, (lane c) S100A6, (lane d)S100A4, (lane e) S100A2 and (lane f) S100A3 (2 µg of each protein) wasapplied. B: Western blot analysis with antiserum against S100A6(1:5,000). The antiserum recognized only S100A6 and no other S100proteins. C: SDS-PAGE; (left lane) one of the total rat brain extracts(100 µg of protein) was applied. Western blot (right lane) with antiS100A6 serum (diluted 1:800); arrow indicates the specific reactionwith S100A6 (dimer). Upper and lower arrowheads indicate the topand the front of the gel, respectively.

S100A6 DISTRIBUTION IN THE RAT NERVOUS SYSTEM 237

Figure 2

Fig. 2. A–I: Camera lucida drawings of representative sectionsshowing the distribution of S100A6-IR structures in the rat centralnervous system (CNS). Transverse sections. The S100A6-IR astrocyteswere mapped to the left halves and S100A6-IR neurons, nerve fibers,

and terminals were mapped to the right halves. The distances fromthe bregma (br) as well as the nomenclature were mostly according tothe atlas of Paxinos and Watson (1997).

Figure 3


parvalbumin (1:10,000; Kagi et al., 1987); and 5) mouse-monoclonal anti-recombinant rat calbindin D-28k (1:10,000; Pinol et al., 1990).

Sections were then washed in PBS three times for 15–20minutes each and incubated for 2–4 hours in eitherbiotinylated donkey anti-goat IgG (dilution 1:200; Amer-sham, Buckinghamshire, England), goat anti-rabbit IgG(dilution 1:200; Vector, Burlingame, CA), or horse anti-mouse IgG (dilution 1: 200; Vector) in 1% BSA in 0.1 M PBScontaining 0.3% Triton X-100. Sections were washed simi-larly and then incubated in avidin-biotin complex (ABC,dilution 1:200; Vector) in 0.1 M PBS containing 0.1%Triton X-100 for 1.5–3 hours at room temperature, accord-ing to the method of Hsu et al. (1981). After rinsing in PBSfollowed by 50 mM Tris buffer (pH 7.6), sections wereincubated in 0.05% diaminobenzidine tetrahydrochloride(DAB; Sigma, St. Louis, MO) in 50 mM Tris buffer contain-ing 0.01% H2O2 for 5–10 minutes at room temperature.After completing the immunohistochemical procedures,the sections were treated for 10 minutes with 0.1% OsO4 in0.1 M phosphate buffer at room temperature to enhancethe reaction products. Finally, sections were washed indistilled water, dehydrated in a graded series of ethanol,infiltrated in propylene oxide, and flat-embedded in Epon-Araldite.

As controls, the antisera against S100A6 and S100Bwere pretreated with corresponding excess antigens (2nmol of purified S100A6 and S100B proteins in 1 ml ofdiluted antisera, respectively). In these controls no specificstaining was detected. To examine the cross-reactivityfurther, the antisera against S100A6 were replaced by theantisera pretreated with 2 nmol of purified S100B and viceversa. These pretreatments had no effect on the immunore-actions.

To reveal the topographical distribution of S100A6-immunoreactive (S100A6-IR) elements in more detail,some sections were counterstained with toluidine blue orcresyl violet after immunohistochemistry. After incubationwith DAB solution, the sections were rinsed with Trisbuffer and then with phosphate buffer, mounted on gelatin-coated glass slides, air-dried, and stained with toluidineblue or cresyl violet.

Fluorescent immunohistochemistryand propidium iodide (PI) staining

Some sections were processed for multiple fluorescentimmunohistochemistry for S100A6 (1:5,000) and one or

two of S100B (1:5,000), GFAP (1:5,000), parvalbumin(1:5,000), and calbindin (1:10,000). Sections were incu-bated in a mixture of primary antibodies in 1% BSA in 0.1M PBS containing 0.3% Triton X-100 and 0.05% sodiumazide for 1–2 weeks at 20°C. For double fluorescentimmunostaining, after rinsing in PBS, they were incu-bated in biotinylated donkey anti-goat IgG (1:200; Jack-son, West Grove, PA) overnight, rinsed, and incubated in amixture of fluorescein isothiocyanate-conjugated streptavi-din (st-FITC, 1:200; Amersham) and Cy5-conjugated don-key secondary antibody, that is, anti-rabbit or anti-mouseIgG (1:500; Jackson) overnight. Sections were furtherstained with propidium iodide (PI, 5 µg/ml in PBS; 30minutes), rinsed, and mounted in Vectashield (Vector). Fortriple fluorescent immunostaining, sections were incu-bated in a mixture of goat polyclonal anti-S100A6, rabbitpolyclonal anti-S100B, and mouse monoclonal anti-GFAP,or a mixture of goat polyclonal anti-S100A6, rabbit poly-clonal anti-parvalbumin, and mouse monoclonal anti-calbindin for 1–2 weeks at 20°C. After incubation inbiotinylated donkey anti-goat IgG overnight, sections wererinsed several times in PBS and incubated in a mixture ofLissamine rhodamine-conjugated streptavidin (st-LRSC)(1:250; Molecular Probes, Eugene, OR), Cy5-conjugateddonkey anti-mouse IgG, and FITC-conjugated donkeyanti-rabbit IgG (1:200; Jackson) overnight. After rinsingseveral times in PBS, sections were mounted in Vecta-shield.

Production of polyclonal antibody againstrecombinant human S100A6, SDS-PAGE,

and Western blot analysis

The human cDNA of S100A6 was introduced into theprokaryotic expression vector pGEMEX (Promega, Madi-son, WI). The vector was transformed into BL-21Lys S cellsand after induction of the bacterial cultures with isopropyl-b-D-thiogalactopyranoside (IPTG), S100A6 was extractedand purified to homogeneity as described previously (Ped-rocchi et al., 1994). Polyclonal antiserum against recombi-nant human S100A6 was raised in goat, and its specificitywas proved by Western blot analysis (Ilg et al., 1996a).Whole rat brains were crushed in liquid nitrogen and thefine powder was suspended in extraction buffer (20 mMTris, pH 7.4; 0.3 M KCl; 10% glycerol; 1 mM EDTA; 1 mMphenylmethanesulphonyl fluoride (PMSF); 2 mM dithio-threitol (DTT); 5 µg/ml leupeptine). The suspension waspelleted twice by centrifugation (10,000g, 15 minutes at4°C), and the protein concentration of the supernatant wasdetermined spectrophotometrically.

Total rat brain extracts and purified recombinant S100proteins were first separated on Tricine sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)(running gel, 15%; stacking gel, 3%) under reducing condi-tions (loading buffer containing 0.5% b-melcaptoethanol[bME]), then blotted onto 0.2 µm nitrocellulose (Schleicherand Schuell, Dassel, Germany), and finally cross-linked byUV radiation. Proteins were visualized by Ponceau Sstaining. The membrane was blocked overnight in 3% BSAand 1% fetal calf serum (FCS), and then incubated withantiserum against S100A6 (diluted 1:800) for 2 hours.Afterward the membrane was incubated with horseradishperoxidase-coupled rabbit anti-goat IgG (1:3,000; Sigma)for 1 hour. The bands were visualized using a premixedsubstrate kit (catalog no. 170–6431; Bio-Rad) following themanufacturer’s instructions.

Fig. 3. A: Light micrograph of an S100A6-immunostained 50-µm-thick coronal section of the olfactory bulb corresponding to Figure 2A.B: Higher magnification image of the boxed area in A. S100A6-IRastrocytes are seen in the subependymal layer around the olfactoryventricle (E/OV) and around the accessory olfactory bulb (AOB). In theglomerular layer (Gl) some glomeruli are S100A6-IR. C: Parasagittalsection (50 µm in thickness) of the main olfactory bulb. Glomerulishow various intensity of S100A6 immunoreactivity. D: Higher magni-fication image of the upper boxed area D in C. Near the border betweenthe Gl and external plexiform layer (EPl) some S100A6-IR astrocytes(arrows) are scattered. E: Higher magnification image of the middleboxed area E in C. In the olfactory nerve layer (ON) intenselyS100A6-IR varicose nerve fibers are seen. F: Higher magnificationimage of the lower boxed area F in C. S100A6-IR varicose nerve fibersgather to S100A6-positive nerve bundles of various sizes and enter theS100A6-IR glomeruli. G: Confocal laser scanning microscopic projec-tion image (about 4 µm in thickness) of a S100A6-IR olfactory receptorcell. Arrow indicates the axon. Scale bars 5 0.5 mm in A–C, 100 µm inD–F, 20 µm in G.


RESULTS

Western blotting

Immunoblot analysis was performed to confirm theantibody specificity (Fig. 1A,B) and the nature of S100A6immunoreactivity in the adult rat brain (Fig.1C). Asshown in Figure 1A and B, the antiserum used in thepresent study recognized only S100A6 protein and did notshow any cross-reactivity against other S100 proteins.Furthermore, the Western blot analysis revealed that inthe rat brain our antibody recognized specifically only oneband at an approximate molecular weight of 21 kDa,corresponding to the dimer of S100A6 protein (Fig.1C).

General remarks on immunostaining

In the present study we recognized no appreciabledifferences between samples fixed by different fixatives inthe immunostaining. In general, the intensity of immuno-staining of S100A6-IR elements varied from strong tofaint. In the present study we intended to show a generalpattern of S100A6-IR elements throughout the rat nervoussystem and therefore we mainly described the distributionand structural features of the intensely stained S100A6-IRelements.

Figure 2 summarizes the distribution of S100A6-IRelements in the rat CNS. Although S100A6-IR elementswere encountered throughout the CNS, they were usuallyclustered in the olfactory bulb, in the white matter of theforebrain, around the ventricles, aqueduct, and centralcanal, in some regions of the limbic system, and in somespecific tracts of the hindbrain and the spinal cord. Fourdifferent types of S100A6-IR elements were found through-out the rat CNS; these were 1) neuronal somata anddendrites; 2) nerve fibers and axon terminals; 3) astrocytesthat were confirmed by double- and triple-labeled fluores-cent immunohistochemistry for S100A6, S100B, and GFAP;all S100A6-IR glial cells were also S100B positive but somewere GFAP negative; and 4) ependymal cells, especiallyaround the central canal .

Olfactory bulb and olfactory mucosa

In the main olfactory bulb as well as in the accessoryolfactory bulb, prominent S100A6-IR elements were olfac-tory nerves in the olfactory nerve layer and some glo-meruli, some glial cells in the subependymal layer, andsome ependymal cells around the olfactory ventricle (Fig.3A,B). However, throughout layers, no intrinsic neuronsbut a few scattered astrocytes were S100A6-IR.

In the olfactory nerve layer of the main olfactory bulb,S100A6-IR varicose nerve fibers and bundles with variousintensity were intermingled (Fig. 3C,F). We then exam-ined the olfactory mucosa to search for the origins of theseS100A6-IR olfactory nerve fibers, and identified S100A6-IRolfactory receptor cells; basal cells and supporting cellswere negative (Fig. 3G).

In the glomerular layer, a few small intensely S100A6-IRglomeruli were scattered among moderately positive,slightly positive, and negative glomeruli (Fig. 3A–D). Insome sections, several intensely S100A6-IR nerve bundlesand fibers were seen to converge on single intenselyS100A6-IR glomeruli (Fig. 3F). Although the intensity ofthe S100A6 immunoreactivity varied considerably amongglomeruli, it appeared to be homogeneous in individualglomeruli, that is, all olfactory nerve fibers and terminals

entering into a glomerulus appeared to be homogeneous intheir S100A6 immunoreactivity, presumably reflecting thehomogeneity of primary olfactory receptors converging onan individual glomerulus (Ressler et al., 1994; Vassar etal., 1994; Mombaerts et al., 1996).

In the accessory olfactory bulb a few scattered S100A6-IRastrocytes were seen (Fig. 3B). A few S100A6-IR varicosenerve fibers and terminals were also encountered in theglomerular layer (Fig. 4B), although the number ofS100A6-IR glomeruli in the accessory olfactory bulb wassmall compared with that in the main olfactory bulb.

As we reported previously (Yamashita et al., 1997), inthe core of the bulb S100A6-IR astrocytes and/or ependy-mal cells were clustered, which extended to the ependymaland subependymal layers of the lateral ventricle, forminga cell cord, that is, ‘‘the rostral migratory stream’’ (Fig. 4A).In addition, some S100A6-IR astrocytes were scatteredaround these S100A6-IR cell clusters at the granular celllayer of the main olfactory bulb. When consecutive sec-

Fig. 4. A: Light micrograph of an S100A6-immunostained 50-µm-thick parasagittal section showing ‘‘the rostral migratory stream.’’ Astrand of S100A6-IR elements extends from the left ventricle (not seenin this figure) to the main olfactory bulb (MOB). B: Higher magnifica-tion image of the boxed area in A. Many S100A6-IR astrocytes areobserved in the subependymal layer around the olfactory ventricle(E/OV). Arrowheads indicate S100A6-IR glomeruli (from the vomero-nasal nerve), and arrows indicate S100A6-IR astrocytes in the acces-sory olfactory bulb (AOB). C: Higher magnification image of the boxedarea in B. S100A6-IR astrocytes radiate several processes withnumerous fine irregular appendages parallel to the rostral migratorystream. Scale bars 5 1 mm in A, 500 µm in B, 100 µm in C.


Fig. 5. Confocal laser scanning microscopic projection images,fluoroscently triple immunostained for GFAP (1), S100A6 (2), andS100B (3). Arrows indicate some representative triple labeled cells.A: Parasagittal section of the forceps minor of the corpus callosum(about 13 µm in thickness). B: Coronal section of the border betweenthe cingulum (upper right) and the corpus callosum (lower left) (about

8 µm in thickness). C: Parasagittal section of the fimbria (about 19 µmin thickness). Besides the S100A6-IR astrocytes, some nerve bundles(arrowheads) are S100A6-IR in the fimbria (C2). D: Coronal section ofsubventricular region of the lateral ventricle (LV) (about 3 µm inthickness). All ependymal cells are S100B-IR (D3). Scale bar 5100 µm.

Figure 6


tions were stained with antibodies against S100B andGFAP, or examined with triple labeling fluorescent immuno-histochemistry, numerous S100B-IR and GFAP-IR cellswere scattered throughout the layers of the main olfactorybulb, in prominent contrast to the restricted distribution ofS100A6-IR astrocytes (see Figs. 2 and 3 in Yamashita et al.[1997]). In individual astrocytic processes, S100A6 immu-noreactivity was generally seen up to more distal portionsthan the S100B immunoreactivity. In the subependymallayer around the olfactory ventricle the somata of theS100A6-IR astrocytes were small and flattened or elon-gated parallel to the layers. S100A6-IR processes arisingfrom these somata were also spread out parallel to thelayers (Figs. 3B, 4C).

These observations indicated that the S100A6-IR cellsin the subependymal layer around the olfactory ventriclewere a particular subpopulation of astrocytes. In additionto astrocytes we also observed some S100A6-IR ependymalcells, which were strongly S100B-IR (Yamashita et al.1997).

Glial cells in the subventricular zone

In addition to the rostral migratory stream (describedabove), some S100A6-IR astrocytes were observed in thesubventricular zone surrounding the lateral ventricle (Figs.5D, 6A), third ventricle (Fig. 6F), aqueduct (Fig. 9A),fourth ventricle (Figs. 9B, 10A), and central canal of thebrainstem (Fig. 10B–D). However, around the centralcanal of the spinal cord, no S100A6-IR glial cells wereobserved (Fig. 10E). In several particular regions such asthe lateral septal nucleus (Fig. 6A), the ventral part of theparaventricular hypothalamic nucleus (Fig. 6F), the cen-tral gray (Fig. 9A), the medial vestibular nucleus (Fig. 9B),and the dorsal motor nucleus of vagus (Fig. 10B,C),S100A6-IR astrocytes extended to the deeper parts of thesubventricular zone or into the surrounding gray matter.S100A6-IR astrocytes were also scattered near the hippo-campal fissure (Fig. 7C).

Ependymal cells

Ependymal cells were usually S100B-IR, whereas only afew ependymal cells were also S100A6-IR. Around the

lateral ventricle, third ventricle, aqueduct, and fourthventricle, some of S100A6-IR ependymal cells were ob-served to extend basal processes; they were assumed to betanycytes (Fig. 6D). At the central canal in the brainstemand the spinal cord at all levels, however, a relatively largenumber of S100A6-IR ependymal cells (including tany-cytes) were observed (Fig. 10G). Thus a subpopulation ofependymal cells including tanycytes was considered to beS100A6-IR.

Glial cells in the white matter

Numerous S100A6-IR glial cells were seen in the whitematter such as the corpus callosum, internal and externalcapsules, and cingulum. A moderate to small number ofS100A6-IR glial cells were encountered in the anteriorcommissure, lateral olfactory tract, optic tract, pyramidaltract, nerve bundles in the striatum (Fig. 6E) and globuspallidus, alveus of the hippocampus, fimbria-fornix, hippo-campal commissure, stria terminalis, cerebellar peduncle,and spinal cord. So far, S100A6-IR glial cells were rarelyencountered in the white matter of the midbrain and thehindbrain, except for a few positive cells in the cerebellarpeduncle and pyramidal tract. Their somata were usuallysmall in size and either round or polygonal in shape,extending many processes with lamellar- and leaflet-likeappendages. These S100A6-IR glial cells were furtherconfirmed to be S100B-IR and GFAP-IR by multiple-fluorescent immunohistochemistry, indicating that theywere astrocytes in the white matter (Fig. 5A–C). However,S100A6-IR astrocytes were only a subpopulation of astro-cytes in the white matter, where many S100A6 negativeastrocytes were also seen. Interestingly, S100A6-IR astro-cytes showed some prominent regional differences in theirdistribution.

In the lateral olfactory tract, S100A6-IR astrocytes andtheir processes were scattered mainly in its outer half (Fig.6A). In the anterior commissure, S100A6-IR astrocyteswere rather numerous in the posterior part of the anteriorcommissure, but rare in its intrabulbar part, anterior part,and substantial commissural part (Fig. 6A). In the corpuscallosum, the medial part contained only a few S100A6-IRastrocytes, whereas the lateral part, including the forcepsminor (Fig. 4A) and forceps major (Fig. 7C), containedmany S100A6-IR astrocytes (Fig. 6A,B). ParticularlyS100A6-IR astrocytes swarmed around the border be-tween the corpus callosum and the neighboring structures,such as the cingulum (Figs. 5B, 6B), striatum (Fig. 6A),and alveus (Fig. 7B). Similarly, in the cingulum and in theexternal capsule, many S100A6-IR astrocytes (Figs. 5B,6B) were crowded near the margin of the neighboringstructures such as the corpus callosum, alveus, and cere-bral cortex.

The fimbria (Figs. 5C2, 6F) and alveus of the hippocam-pus and the ventral hippocampal commissure (Fig. 6A,C)contained S100A6-IR astrocytes as well as severalS100A6-IR nerve bundles. As for the fornix, the S100A6-IRastrocytes were rare (Fig. 6A), except for the caudal part ofthe dorsal fornix, where several S100A6-IR astrocyteswere encountered (Fig. 6A).

In the spinal cord, S100A6-IR astrocytes were mainlydistributed in the outer part of the white matter (Fig. 10E).One or two processes emanated from the immunoreactivesomata, extending radially and spanning the outer two-thirds of the white matter from the pial surface (Fig. 10H).These distribution patterns showed no prominent differ-

Fig. 6. A: Light micrograph of a 50-µm-thick S100A6-immuno-stained coronal section corresponding to Figure 2C. B: Higher magni-fication image of the border between the cingulum (cg) and corpuscallosum (cc), containing many S100A6-IR astrocytes. C: Highermagnification image of the upper left boxed area C in A. SomeS100A6-IR nerve fibers and bundles and an S100A6-IR astrocyte areobserved in the ventral hippocampal commissure (vhc). D: Highermagnification image of the lower boxed area D in A. An S100A6-IRependymal cell with its ependymal fiber (tanycyte) around the thirdventricle is observed in this section. E: Higher magnification image ofthe right boxed area E in A. Some S100A6-IR astrocytes are seenamong nerve bundles in the striatum. F: Light micrograph of anS100A6-immunostained 50-µm-thick coronal section corresponding toFigure 2D. In the hypothalamic nuclei surrounding the third ventricle(3V), e.g., the ventral part of the paraventricular hypothalamicnucleus (PaV), some S100A6 astrocytes are seen. G: Higher magnifica-tion image corresponding to the lower boxed area G in F. In thebasolateral amygdaloid nucleus (BL) some neurons are stronglyS100A6-IR. Faintly S100A6-IR neuronal somata are also scattered.H: Light micrograph of a strongly S100A6-IR neuron and theirdendritic branches in the basolateral amygdaloid nucleus. Arrow-heads indicate dendritic spines. Scale bars 5 1 mm in A and F, 200 µmin B and G, 50 µm in C–E and H.


Fig. 7. A and B: Light micrographs of two S100A6-immunostained50-µm-thick coronal sections showing ventral parts of the hippocam-pal formation corresponding to Figure 2E and F, respectively. A: ManyS100A6-IR neurons are seen in the ventral tip of the CA1-subicular (S)border region, whereas some S100A6-IR neurons are seen in theamygdalohippocampal area (AHi), deep layer of the lateral entorhinalcortex (lEnt), and perirhinal cortex (PRh). B: The parasubiculum(PaS) is strongly S100A6-IR, from which some S100A6-IR nerve fibersenter the angular bundle (ab). S100A6-IR neurons are scatteredin the deep layers of the lEnt. C: Light micrograph of an S100A6-

immunostained 50-µm-thick parasagittal section showing the hippo-campal formation. Arrowheads indicate S100A6-IR presumed pyrami-dal cells at layer 5 in the dorsal portion of the medial entorhinal cortex.The hippocampal formation displays the dorsoventral gradient in theS100A6 immunoreactivity. D: Higher magnification image of theboxed area in A, showing the S100A6-IR neurons in the ventral tip ofthe CA1-subicular border region and the AHi. E: Higher magnificationimage of the boxed area in D. An S100A6-IR pyramidal neurondisplays spines (arrowheads) on its dendritic branch. Scale bars 5 1mm in A–C, 200 µm in D, 20 µm in E.


ence among various levels, that is, the cervical, thoracic,lumbar, and sacral levels.

In the gray matter, a few isolated S100A6-IR protoplas-mic astrocytes, which had larger cell bodies and morearborized processes than those in the white matter de-scribed above, were encountered (Fig. 9C).

Neurons in the limbic system

S100A6-IR neurons were observed in some particularregions of the limbic system in the rat brain; they wereclustered in the ventral tip of the CA1-subicular borderregion (see Discussion), parasubiculum, entorhinal cortex,and basolateral amygdaloid nucleus, whereas someS100A6-IR neurons were scattered in various areas in thelimbic system (Fig. 7C). In addition, S100A6-IR nervefibers and terminals were also seen in various regions inthe limbic area.

Amygdala. S100A6-IR neurons were clustered in thebasolateral amygdaloid nucleus, some of which were

strongly positive and some faintly positive (Fig. 6G).Strongly S100A6-IR neurons had large somata with slen-der dendritic processes (Fig. 6H), which sometimes showedapparent dendritic spines, indicating that they were mainlypyramidal cells (McDonald, 1982, 1984, 1992), whereas itwas difficult to analyze dendritic features of faintlyS100A6-IR neurons. In addition to somata and dendriticprocesses, scattered S100A6-IR varicose nerve terminalswere detected in the basolateral amygdaloid nucleus.

Since parvalbumin and calbindin immunoreactive neu-rons were reported to be clustered in this region (Celio,1990; McDonald, 1996), we examined the possible colocal-ization of these three calcium-binding proteins in someneurons using the triple fluorescent immunostainingmethod. Strongly S100A6-positive neurons showed nei-ther parvalbumin nor calbindin immunoreactivity, whereassome faintly S100A6-positive neurons were parvalbuminand/or calbindin immunoreactive (data not shown).

In the amygdalohippocampal area, strongly to faintlyS100A6-IR neurons were encountered (Fig. 7A,D), some ofwhich also showed dendritic spines. In addition, someS100A6-IR varicose nerve terminals were observed in thisregion.

Hippocampus and subiculum. In the ventral tip ofthe CA1-subicular border region, but neither in the middlenor in the dorsal level, many strongly S100A6-IR neuronalsomata were encountered in the pyramidal cell layer (Fig.7A,C). Strongly S100A6-IR neurons were triangular, ovoid,or pyramidal in somal shape. They extended one thickapical dendrite and several basal dendrites, and spineswere occasionally seen on these dendritic branches (Fig.7D,E); most of these S100A6-IR cells were assumed to bepyramidal cells. Moreover, in the strata lacunosum-moleculare, pyramidale, and oriens at and near this borderregion, S100A6-IR varicose nerve fibers and terminalswere seen, some of which ran along each stratum, whichprobably contained associational and/or afferent axon ter-minals (see discussion with Tables 1 and 2). A few scat-tered S100A6-IR neurons were also seen in the CA1 region(Fig. 8A), but, so far, we encountered no S100A6-IRneuronal somata in the dentate gyrus and CA3/CA2region.

Many S100A6-IR nerve fibers were seen in the ventralportion of the alveus (Fig. 8G), but in the dorsal portion nodistinctly S100A6-IR nerve fibers were encountered. In thefimbria of the hippocampus (Fig. 6F) and the ventralhippocampal commissure (Fig. 6A,C), S100A6-IR nervefibers were gathered and made up thick S100A6-IR nervebundles. On the other hand, no distinctly S100A6-IR nervefibers were seen in the fornix (Fig. 6A), dorsal fornix, anddorsal hippocampal commissure.

Parasubiculum and presubiculum. In the presentdescription we virtually follow Blackstad’s definition (1956)of three subicular subdivisions and laminar organizationsof the subicular complex. The parasubiculum showed aprominently intense S100A6 immunoreactivity at the ven-tral level. In layers 2 and 3 of the ventral parasubiculum,intensely S100A6-IR neurons and their processes weredensely clustered (Figs. 7B, 8A,B), whereas in layer 1,S100A6-IR nerve fibers and terminals were intermingledso densely that, at low magnifications (Fig. 8A,B), thislayer could not be discriminated clearly from layers 2 and3. In a slightly more dorsal portion of the ventral parasu-biculum, where the intensity of the S100A6 immunoreac-tivity decreased slightly, S100A6-IR neuronal somata and

TABLE 1. Summary of the S100A6-IR Efferent Projectionsin the Lymbic System

S100A6-IR neuronsPresumed

target regions Reference1

Ventral tip of CA1-subicular Subiculum (S) 1border region Parasubiculum (PaS) 1

Entorhinal cortex (Ent) 1, 2Lateral septal nucleus (LS) 1, 2Basolateral amygdaloid nucleus

(BL)1–3

Bed nucleus of the stria terminalis(BST)

2, 3

Parasubiculum (PaS) Bilateral entorhinal cortex (Ent) 4–7Contralateral parasubiculum

(PaS)4

Medial entorhinal cortex (mEnt)Layer 2 Dentate gyrus (DG) 8, 9Layer 4, 5 Superficially directed intra-

entorhinal area8

Parasubiculum (PaS) 8Lateral entorhinal cortex (lEnt)

layer 6Layers 4–6 of the mEnt 10

Basolateral amygdaloid nucleus(BL)

Bed nucleus of the stria terminalis(BST)

Ventral subiculum (S)

11–13

11Amygdalohippocampal area (AHi) Ventral CA1/subiculum (S) 14

1References as follows: 1, van Groen and Wyss, 1990b; 2, Gronewegen et al., 1987; 3,Canteras and Swanson, 1992; 4, van Groen and Wyss, 1990a; 5, Kohler, 1985; 6,Caballero-Bleda and Witter, 1993; 7, Caballero-Bleda and Witter, 1994; 8, Kohler, 1986;9, Witter, 1993; 10, Kohler, 1988; 11, Krettek and Price, 1978; 12, de Olmos et al., 1985;13, McDonald, 1991; 14, Canteras et al., 1992.

TABLE 2. Summary of the S100A6-IR Afferent Projectionsin the Lymbic System

S100A6-IR nervesand/or terminals Presumed origins Reference1

Ventral tip of CA1-Subicular CA1 1border region Amygdalohippocampal area (AHi) 2

Basolateral amygdaloid nucleus (BL) 3Parasubiculum (PaS) 4Layer 3 of the entorhinal cortex (Ent) 5

Parasubiculum (PaS) Basolateral amygdaloid nucleus (BL) 4CA1/subiculum (S) 4Contralateral parasubiculum (PaS) 4Entorhinal cortex (Ent) 6

Medial entorhinal cortex (mEnt)Layer 2 Contralateral parasubiculum (PaS) 7Layer 4 CA1/subiculum (S) 8, 9

Basolateral amygdaloidnucleus (BL)

Ventral CA1Ventral subiculum (S)

92, 8

Amygdalohippocampalarea (AHi)

Ventral CA1/subiculum (S) 10

1References as follows: 1, Amaral et al., 1991; 2, Canteras and Swanson, 1992; 3, Krettekand Price, 1977; 4, van Groen and Wyss, 1990a; 5, Caballero-Bleda and Witter, 1994; 6,Kohler, 1986; 7, Kohler, 1985; 8, Gronewegen et al., 1987; 9, van Groen and Wyss, 1990b;10, Canteras et al., 1992.


Figure 8


spiny dendrites were clearly distinguished (Fig. 8H), indi-cating that they were pyramidal cells. Among S100A6-positive and -negative neurons, S100A6-IR varicose nervefibers and puncta were also encountered. Axonal processesarising from S100A6-IR somata could sometimes be tracedto the angular bundle (Figs. 7B, 8B). Furthermore, in theventral portion of these parahippocampal regions,S100A6-IR varicose nerve fibers from the parasubiculumwere traced to the medial entorhinal cortex. ThusS100A6-IR neurons in the parasubiculum were assumedto contain projection neurons in this region. The triangulartransition area in the parasubiculum, located between aradial bulge of the lamina dissecans and the medialentorhinal cortex (Fujise et al. 1995), was intensely calbin-din immunoreactive but S100A6 negative except for a fewscattered strongly S100A6-IR neurons (Figs. 7B, 8D). Onlya few strongly S100A6-IR neurons were scattered in thepresubiculum (Fig. 8B). Therefore, the S100A6 immunore-activity appeared to be complementary to the calbindinimmunoreactivity in these parahippocampal regions (Fig.8E,F). In the more dorsal portion, S100A6-IR elementswere decreased in number and only a few positive ele-ments were scattered (Fig. 7C), showing a dorsoventralgradient of the S100A6 immunoreactivity in the parasu-biculum.

Entorhinal cortex. The terminology of the layers inthe entorhinal cortex adopted in this study is based on thatof Lorente de No (1933), that is, layer 1: the molecularlayer; layer 2: the stellate cell layer; layer 3: the superficialpyramidal cell layer; layer 3a: a cell-poor zone also calledthe lamina dissecans; layer 4: the deep pyramidal celllayer; layer 5: the small pyramidal cell layer; and layer 6:the polymorph cell layer. In the medial entorhinal cortexlayers 2 and 4 were more intensely S100A6-IR than otherlayers, showing a prominent band pattern resembling thatof the calbindin immunoreactivity. Strongly to moderatelyS100A6-IR neurons were scattered mainly in layer 2, the

superficial part of layer 3, and layer 4 (Fig. 8B). In layers 2and 4, S100A6-IR nerve fibers and puncta were clustered(Fig. 8C), although some varicose S100-IR nerve fiberswere seen running all over the layers of the medialentorhinal cortex. Several S100A6-IR nerve fibers weretraced to the angular bundle (Fig. 8B) and to the parasu-biculum. Corresponding to the S100A6-IR neurons in themedial entorhinal cortex, S100A6-IR fibers and terminalswere encountered in the middle molecular layer of thedentate gyrus (Fig. 8I), the target zone of layer 2 projectionneurons. At the more lateral portion of the medial entorhi-nal cortex, the S100A6 immunoreactivity decreased (Fig.8A). In addition, at the dorsal portion but not at theventral portion of the medial entorhinal cortex, a fewstrongly S100A6-IR neurons were encountered at layer 5,which had large somata, ovoid or pyramidal in shape,extended apical dendrites to the superficial layers, andsometimes were seen to have dendritic spines; theseS100A6-IR neurons were assumed to be pyramidal cells(Fig. 7C).

In the lateral entorhinal cortex, on the other hand,strongly to faintly S100A6-IR neurons were scatteredmainly at layer 6b, the deepest layer of the medialentorhinal cortex (Fujimaru and Kosaka, 1996) (Fig. 8G);some of them showed dendritic spines. Moreover,S100A6-IR varicose nerve terminals were also found there.In the deep layer of the perirhinal cortex adjacent to thisregion, a few moderately to faintly S100A6-IR neuronswere encountered (Fig. 7A,B).

Other regions. In the lateral mammillary nucleus, afew lightly stained S100A6-IR neurons were observed,although neither S100A6-IR neurons nor S100A6-IR nervefibers and terminals were encountered in other parts of themammillary body (not shown).

Scattered S100A6-IR varicose nerve terminals wereseen in the lateral septal nucleus (Fig. 6A), and a fewS100A6-IR varicose nerve terminals were found in the bednucleus of the stria terminalis as well.

Sensory tracts and neurons

In the brainstem and spinal cord, some distinct S100A6immunoreactive nerves and axon terminals but no neuro-nal somata were observed.

The sensory root of the trigeminal nerve (Fig. 9A,D) andthe spinal trigeminal tract (Figs. 9B, 10A–D) containedmany S100A6-IR nerve fibers. In the spinal trigeminaltract, the outer (lateral) half was occupied by S100A6-IRfibers, whereas the inner (medial) half was mainly occu-pied by S100A6-negative nerve fibers with sparsely scat-tered positive ones (Fig. 9F). In the spinal trigeminal tract,the paratrigeminal nucleus contained S100A6-IR nerveterminals (Fig. 10B).

In the dorsomedial part of the principal sensory trigemi-nal nucleus and the dorsomedial spinal trigeminal nucleus,several clusters of S100A6-IR fibers and terminals weresparsely scattered (Fig. 9E). In the interpolar part of thespinal trigeminal nucleus, S100A6-IR nerve fibers pen-etrated from the dorsolateral part to the medial part andcomposed plexus-like clusters of terminals at the marginsof the neighboring structures (Fig. 10B,C), e.g., the spinaltrigeminal tract, parvicellular reticular nucleus, and cau-dal part of the spinal trigeminal nucleus. In the caudalpart of the spinal trigeminal nucleus, the marginal zone(lamina 1) and the gelatinous part (lamina 2) had dense

Fig. 8. A: Light micrograph of an S100A6-immunostained 50-µm-thick horizontal section showing the ventral part of the hippocampalformation. B: Higher magnification image of the boxed area B in A,showing S100A6-IR components, the parasubiculum (PaS) and medialentorhinal cortex (mEnt). Some S100A6-IR axonal processes from thePas are traced to the angular bundle (ab). C: Higher magnificationimage of the left boxed area C in B, showing some S100A6-IR neuronalfibers and puncta in layer 2, and an S100A6-IR presumed pyramidalneuron in the superficial part of layer 3. D: Higher magnificationimage of the right boxed area D in B. In the triangular transition area(Tr), a few scattered S100A6-IR neurons are observed. E,F: Lightmicrographs of consecutive horizontal sections (50 µm in thickness)about 400 µm dorsal to A. The S100A6 immunoreactivity (E) appearscomplementary to the calbindin immunoreactivity (F) in the Tr, PaS,and presubiculum (PrS). G: Higher magnification image of the leftboxed area G in A. S100A6-IR neurons are scattered in the deepestlayer (layer 6b) of the lateral entorhinal cortex (lEnt), and S100A6-IRnerve fibers are running in the alveus (alv) through the externalcapsule (ec). Arrows indicate S100A6-IR fiber connecting layer 6b ofthe lEnt and the alv. H: Higher magnification image of the boxed areain E. S100A6-IR neuronal dendrites with spines (arrowheads) areobserved in the parasubiculum. I: Light micrograph of an S100A6-immunostained 50-µm-thick coronal section showing the molecularlayer (ML) and granular layer (GL) of the dentate gyrus. S100A6-IRfibers and terminals are observed in the middle ML, presumablycorresponding to axon terminals of S100A6-IR neurons in the medialentorhinal cortex. Scale bars 5 1 mm in A, 200 µm in B, E, F, and I, 50µm in C, D, and G, 20 µm in H.


Figure 9


S100A6-IR fibers and terminals, which entered into theseregions from the spinal trigeminal tract (Fig. 10D). In theinner part of the caudal part of the spinal trigeminalnucleus, that is, the magnocellular layer (laminae 3 and 4),sparse S100A6-IR fibers and terminals were encountered(Fig. 10C). Sparse S100A6-IR nerve fibers and terminalswere also encountered in the cuneate nucleus and gracilenucleus (Fig. 10D), but very few in the external cuneatenucleus. In the area postrema, moreover, some S100A6-IRnerve fibers were observed (Fig. 10B).

The solitary tract also contained numerous S100A6-IRfibers, and in the nucleus of the solitary tract S100A6-IRterminals were scattered (Fig. 10A–D). Moreover,S100A6-IR afferent nerve bundles of cranial nerves, pre-sumably the vagus nerve and/or grossopharyngeal nerve(Beckstead and Norgren, 1979; Sugimoto et al., 1997),were observed to enter into the solitary tract across thespinal trigeminal tract (Fig. 10A,F).

In the spinal cord (Fig. 10E), the distribution pattern ofS100A6-IR elements was conserved throughout the cervi-cal, thoracic, lumbar, and sacral levels. The dorsal rootfibers and the tract of Lissauer (the dorsolateral fascicu-lus) contained S100A6-IR nerve fibers. In the more centralparts, the marginal zone (substantia spongiosa or layer 1)and the substantia gelatinosa (layer 2) contained denseS100A6-IR fibers and terminals. In the proper sensorynucleus of the dorsal horn (layers 3 and 4), sparseS100A6-IR fibers and terminals were encountered. Inaddition, in the posterior funiculus (that is, the cuneatefasciculus and gracile fasciculus), especially in its moresuperficial part, clusters of fine S100A6-IR nerve fibersrunning longitudinally were observed.

We also examined the trigeminal ganglia and dorsal rootganglia to search for the origins of the S100A6-IR fibersand terminals observed in the brainstem and spinal cord.In both ganglia, S100A6-IR primary sensory neurons andtheir processes were encountered. Our double fluorescentimmunostaining (Fig. 11) confirmed that S100A6-IR sen-sory neurons were usually small, whereas S100B-IR sen-sory neurons were mostly large, as reported previously(Ichikawa et al., 1997), although some sensory neurons inboth ganglia contained both proteins. Schwann cells wereboth S100A6-IR and S100B-IR, whereas satellite cellswere S100B-IR but not S100A6-IR. Most myelinated nervefibers surrounded by Schwann’s sheaths were S100B-IRbut not S100A6-IR. Furthermore, presumed unmyelinatednerve fibers, which were S100A6-IR but not S100B-IR,

were encountered in both ganglia. In addition, perineuriaof both ganglia were S100A6-IR but not S100B-IR.

DISCUSSION

S100A6 was reported to be expressed in various cells,such as fibroblasts and epithelial and muscle cells, andoverexpressed in various tumor cells (Engelkamp et al.,1992; Weterman et al., 1992; Ilg et al., 1996a). A previousstudy on the localization of S100A6 in the CNS reportedthat only some neurons but no glial cells in the rat nervoussystem were S100A6-IR (Filipek et al., 1993). However,our present study showed that not only some subpopula-tions of neurons but also some glial cells as well asependymal cells are S100A6-IR.

Specificity of antibody and immunostaining

Our polyclonal antibody was raised in goat againstrecombinant human S100A6 (Ilg et al., 1996a) and cross-reacted neight with other S100 proteins nor with otherCa21-binding proteins. Rat S100A6 is highly homologousto human S100A6 (Murphy et al., 1988), and thus thisantibody is expected to detect S100A6 in the rat brain. Thepresent Western blotting showed that our antibody de-tected only one band of approximate molecular weight 21kDa in the rat brain extract. S100 A6 is a non-covalenthomodimeric protein (molecuar weight of dimer, 21 kDa).The dimeric interface is mediated by strong hydrophobicinteractions between helix IV and helix IV’ (Potts et al.,1995) and not by intermolecular disulfide bridges. In totaltissue extracts the dimer is further stabilized by its stronginteractions with a number of target proteins, making iteven more resistant to dissociation into monomers (Wojdaand Kuznicki, 1994; Schafer and Heizmann, 1996). There-fore, the major form of S100A6 protein in rat brain extractseven under reducing conditions seems to be the dimer.Furthermore, antisera against various S100 proteins seemto be more reactive against the dimeric forms and lessagainst the monomer (personal observations of Heiz-mann). Thus we concluded that the band detected in thepresent Western blotting of the rat brain extract corre-sponded to the dimer of S100A6 proteins.

Apparently our antibody reproducibly stained a substan-tial number of neurons, non-neuronal cells, nerve fibers,and nerve terminals by two different immunohistochemi-cal methods in samples fixed with two different fixatives,one with and the other without a low concentration ofglutaraldehyde. At present the discrepancy between theprevious studies and ours cannot be explained clearly, anddirect comparisons of these two different antibodies arenecessary to understand the possible causes of this discrep-ancy.

S100A6-IR astrocytes and ependymal cells

Compared with the GFAP (Kalman and Hajos, 1989;Hajos and Kalman; 1989) and S100B (Boyes et al., 1986)immunoreactivities, two markers of astrocytes, S100A6immunoreactivity was located only in some subpopula-tions of astrocytes in particular regions. As for S100A6-IRastrocytes in the white matter, two characteristics in theirdistribution can be pointed out. First, S100A6-IR astro-cytes appeared to swarm around the border between theneighboring structures, e.g., between the cingulum and

Fig. 9. A,B: Light micrographs of S100A6-immunostained 50-µm-thick coronal sections showing the pons (A) and the upper part of themedulla oblongata (B, corresponding to Fig. 2G). C: Higher magnifica-tion image of the left boxed area C in A, showing S100A6-IR protoplas-mic astrocytes, which had larger cell bodies and more arborizedprocesses, in the central gray (CG). D: Higher magnification image ofthe right boxed area D in A, showing S100A6-IR nerve fibers in thesensory root of the trigeminal tract (s5) and a few S100A6-IRastrocytes in the middle cerebellar peduncle (mcp). E: Higher magnifi-cation image of the upper boxed area E in B, showing S100A6-IR nervefibers and terminals in the dorsomedial part of the principal sensorytrigeminal nucleus (Pr5DM). F: Higher magnification image of thelower boxed area F in B, showing transversely cut S100A6-IR nervefibers mainly occupying the outer (lateral) half of the spinal trigeminaltract (sp5). Scale bars 5 1 mm in A and B, 50 µm in C–F.


Figure 10


corpus callosum. Second, when they were associated withcommissural fibers, the median part of the commissurescontained only a few S100A6-IR astrocytes, whereas theparamedian and lateral parts contained many S100A6-IRastrocytes, although many S100B-IR and/or GFAP-IRastrocytes were encountered even in the median part.These characteristic localizations of S100A6-IR astrocytesindicate that they might be a particular subpopulation ofastrocytes that play some specific roles in forming, segre-gating, and/or maintaining the nerve fiber bundles andcommissures. Similarly, as we showed previously (Ya-mashita et al., 1997), among the astrocytes at the subepen-dymal layer, S100A6-IR astrocytes at ‘‘the rostral migra-tory stream’’ (Altman, 1969; Luskin, 1993; Lois and Alvarez-Buylla, 1994) represented a particular subpopulation ofastrocytes probably also participating in the formation ofthe rostral migratory stream. Further elucidation of thefunctional roles of S100A6 are needed to get some clues tothe Ca21-dependent functional roles played by those par-ticular S100A6-IR subpopulations of astrocytes and epen-dymal cells.

S100A6-IR neurons in the limbic system

S100A6-IR neuronal somata and axon terminals wereseen in some particular regions of the limbic system,where most or some S100A6-IR neurons appeared to be asubpopulation of pyramidal projection neurons.

Characteristic S100A6-IR pyramidal neurons in theventral part of the hippocampal formation appeared tobelong to both the distal portion of the CA1 region and theproximal portion of the subiculum. Previous tracer studiesalso showed that efferent target regions of these neighbor-ing portions were not completely segregated but ratheroverlapped (Gronewegen et al., 1987; van Groen and Wyss,1990b; Canteras and Swanson, 1992). Therefore we namedthis particular portion the ventral tip of the CA1-subicularborder region.

Taking the previous results of tracer experiments intoconsideration, presumed target regions of S100A6-IR pyra-midal cells, origins of nerve fibers and terminals observedin the present study and possible connections amongS100A6-IR neurons in the limbic system could be summa-rized as shown in Figure 12 and Tables 1 and 2. However, itshould be noted 1) that S100A6-IR neurons were not

necessarily the major subpopulation in each region and 2)that they were assumed to participate in only some partsof the efferent projections from that area, because theknown target regions from such areas (Gronewegen et al.,1987; van Groen and Wyss, 1990a; 1990b; Canteras andSwanson, 1992), for example, the thalamus, striatum, andhypothalamus, contained no S100A6-IR terminals, and inturn the known regions of origins of such areas (van Groenand Wyss, 1990a), for example, the thalamus, contained noS100A6-IR projection neurons (Fig. 12). At any rate, thelimbic regions that contain strongly S100A6-IR neuronsappear to connect with one another directly or indirectly.

S100A6-IR elements in the trigeminaland dorsal root ganglia and primary

sensory pathways

In the dorsal root ganglia (Harper and Lawson, 1985)and trigeminal ganglia (Kai-Kai, 1989), two main groupsof neurons can be distinguished on the bases of size,staining intensity with basic dyes, ultrastructural fea-tures, and chemical properties: large type A neurons andsmall type B neurons. Type A neurons are associated withlow threshold mechanoreceptors, whereas type B neuronsare associated with the nociceptors with unmyelinated orthin myelinated axons. Type B neurons contain excitatoryamino acids, believed to act as neurotransmitters (Batta-glia and Rustioni, 1988; Tracy et al., 1991), and alsoneuropeptides such as calcitonin gene-related peptide,substance P, somatostatin, galanin, and vasoactive intesti-nal polypeptide, which probably serve as neuromodulators(Ju et al., 1987; Kai-Kai, 1989; Smith et al., 1993; Ichikawaet al., 1997). S100A6-IR neurons in both ganglia weresmall in size, and the distributions of the S100A6-IR nervefibers and axon terminals in the brainstem and spinal cordcorrespond well to those originating from type B neuronsin both ganglia (Sugiura et al., 1986; Patterson et al., 1989,1990). Furthermore, our personal observations also sup-port this identification. First, our electron microscopicanalysis confirmed that these S100A6-IR elements wereeither unmyelinated or thin myelinated fibers, but notthick myelinated ones. Second, we confirmed thatS100A6-IR sensory neurons in the ganglia as well as tractsin the brainstem and spinal cord showed the colocalizationof calcitonin gene-related peptide-like immunoreactivity.Thus, S100A6 immunoreactivity might be used as a poten-tial chemical marker of type B neurons, or at least somedistinctive subpopulation of type B neurons, in both gan-glia.

It is also worth noting that S100A6 immunoreactivity inthe sensory systems is located only in some specificprimary sensory neurons (as well as their nerve fibers andterminals), including olfactory receptor cells, but neitherin the secondary nor in the higher order neurons in thesensory systems.

Tonini et al. (1991, 1995) reported that the S100A6 geneis active in neuroblastoma cell lines of Schwann cellmorphology, but not in those of neuroblastic morphologyafter treatment by retinoic acid, and that in neuroblasto-mas, S100A6 was detected in stromal cells, cells withcharacteristic Schwann-like differentiation, nerve sheaths,and perineurium, whereas neuroblasts were negative. Weobserved that Schwann cells and perineuria in both gan-glia were S100A6-IR (Fig. 12A1,B1,C1). Thus, our obser-

Fig. 10. A–D: Light micrographs of S100A6-immunostained 50-µm-thick coronal sections of the medulla oblongata at four differentrostro-caudal levels (C corresponding to Fig. 2H). Nerve fibers andterminals in some regions of the brainstem, e.g., dorsomedial spinaltrigeminal nucleus (DMSp5), solitary tract (sol), nucleus of thesolitary tract (Sol), and spinal trigeminal tract (sp5) are S100A6-IR. Afew astrocytes are also S100A6-IR in the inferior cerebellar peduncle(icp) and the ventral parts of the brainstem near the pia mater.E: Light micrograph of an S100A6-immunostained 50-µm-thick coro-nal section of the spinal cord (C6 level) corresponding to Figure 2I.Nerve fibers and terminals and some radial astrocytes are S100A6-IR.F: Higher magnification image of boxed area in A, showing S100A6-IRnerve terminals in the DMSp5. An S100A6-IR nerve bundle, presum-ably from the vagus nerve (10n), enters into the sol. G: Highermagnification image of the boxed area in C, showing S100A6-IRependymal cells around the central canal (CC). H: Higher magnifica-tion image of the boxed area in E, showing an S100A6-IR radialastrocyte in the white matter of the spinal cord. Scale bars 5 1 mm inA–E, 50 µm in F and G, 20 µm in H.


vations correspond well with their results obtained in cellcultures and neuroblastomas.

In the present study S100A6 has been shown to belocalized in various types of cells in the rat nervoussystem. Its distribution pattern is very unique and consid-erably different from that of other calcium-binding pro-

teins such as parvalbumin (Celio, 1990), calbindin (Celio,1990), calretinin (Rogers, 1991; Jacobowitz and Winsky,1991; Arai et al., 1991), and S100B (Rickman and Wolff,1995). This diverse localization of S100A6 should be takeninto consideration in future analyses of the functionalroles of S100A6 in the CNS.

Fig. 11. Confocal laser scanning microscopic projection images ofthe trigeminal (A) and the dorsal root ganglia (B,C) (about 2 µm inthickness), double-immunostained for S100A6 (1), and S100B (2).S100A6-IR sensory neurons are usually small, whereas S100B-IRsensory neurons are mostly large. Schwann cells in these ganglia areboth S100A6-IR and S100B-IR, whereas most myelinated nerve fibers

surrounded by Schwann’s sheaths are S100B-IR but S100A6 negative.Arrowheads indicate S100A6-IR but S100B-negative perineuria. Ar-rows indicate representative S100A6-IR but S100B negative unmyelin-ated nerve fibers. Asterisks indicate representative primary sensoryneuron showing the colocalization of S100A6 and S100B. Large arrowsindicate S100B-IR but S100A6-negative satellite cells. Scale bar 5 100 µm.


ACKNOWLEDGMENTS

The authors thank Ms. S. Suematsu and Ms. A. Oda fortheir secretarial assistance and Ms. M. Killen for criticalreading of theh manuscript. This work was supported by:1) the Japanese Ministry of Education, Science and Cul-ture: Grants-in-aid for General Scientific Research(09480213 to T. K.); 2) grants from the Uehara MemorialFoundation and the Mitsubishi Foundation to T. K.; 3) theWilhelm Sander-Stiftung (FRG) to C.W.H.; and 4) theBiomed 2, Switzerland (BBW 95.0215–1 to C.W.H.)

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distribution of a specific calcium-binding protein of the s100 protein family, s100a6 (calcyclin),...

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