Journal of Cell Science ihs osberlsi euaigse cell migration stem and regulating proliferation in roles cell possible stem follicle niches: whisker in tenascin-W and Tenascin-C Report Short stems eetycaatrzdmme ftetnsi gene tenascin the of member characterized and recently Tenascin- most 2001). the 1991) (Tucker, is by migration W surrounds McKay, their expression inhibits crest and also tenascin-C neural (Chiquet- the (Tucker of stroma Tenascin-C cells knockdown tumor morpholino-mediated 2012). crest solid neural Tucker, with migrating adult and the Ehrismann in the and in motility embryo, cell and interactions epithelial–mesenchymal with non- and 2008). neurons al., into et Amoh differentiate 2006; to cells potential neuronal the 2008), have Hu, and associated and Sieber-Blum and 2004; bulge al., et whisker and (Sieber-Blum the isolated trabeculae explanting be by can the culture cells of These in 2010). development studied al., the et to (Amoh the shaft contribute to whisker sheath they root where outer papilla, the nestin of cells dermal marker membrane stem cell glassy found CD34-positive the the stem cells along that neural proposed migrate stem been the has of for It CD34. positive population and is second a bulge A is cells the 2010). which near stem sinus, al., venous precursor ring et keratinocyte superficial (Amoh sheathes two keratin-15-positive more root of the whisker by under source hair The all bulge capsule. surrounded a Unlike fibrous in form tough are found a 2010). and follicles organs sinuses (Muchlinski, whisker tactile humans follicles, complex except are mammals follicles Whisker Introduction in role a play might the glycoproteins from these cells. words: cells cells and stem Key stem these tenascin-W, these follicle of and of whisker migration tenascin-C of proliferation the both proliferation and promoting contains the migration in niche increased the glycoproteins nearby cell tenascin-W, directing both these not stem in by but for follicle inhibited niche, formation Tenascin-C, was keratin-15- whisker role The fibronectin bulb. cell CD34-positive a follicles. surrounding on whisker hair with stem cultured region the nearby cells consistent CD34-positive of to trabecular stem is niche niche whisker-derived the cell the which CD34-positive stem in tenascin-W, in in neural complexes the and found and adhesion in tenascin-C focal is found membrane also and tenascin-W glassy is fibers tenascin-C Tenascin-W stress glycoprotein the membrane. glycoprotein of glassy related along the the found follicles, The along whisker we and the whisker cells. immunohistochemistry, trabeculae to membrane Using mouse stem glassy shaft. of the CD34-positive whisker along capsule the negative, bulge of the fibrous near formation the niche the their in in from participate migrate they that cells where stem bulb, CD34-positive has follicle whisker The Summary 10.1242/jcs.134650 doi: 5111–5115 126, ß Science Cell of Journal 2013 September 4 Accepted ( correspondence for *Author 4 3 2 1 Tucker P. Richard aut fSine nvriyo ae,45 ae,Switzerland Basel, 4056 Basel, USA Switzerland Switzerland of 95616, Bern, Basel, University CA 3012 4058 Science, Davis, Bern, Foundation, of Davis, of Research Faculty at University Novartis California Anatomy, Research, of of Biomedical University Institute for Anatomy, Institute Human Miescher and Friedrich Biology Cell of Department 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. eacnCi netaellrmti lcpoenassociated glycoprotein matrix extracellular an is Tenascin-C irsa xrclua arx irnci,C3,Mouse CD34, Fibronectin, matrix, Extracellular Vibrissa, nvitro in [email protected] 1, See-lme l,20;Wn tal., et Wong 2004; al., et (Sieber-Blum ,Jculn Ferralli Jacqueline *, ) 2 oansC Schittny C. Johannes , olce,adw hrceieteefcso eacnCand tenascin-C of effects the cells these characterize on we tenascin-W whisker and mouse in cells follicles, stem it CD34-positive surrounding niche also and the is bone, osteoblasts trabecular 2008). Tenascin-W of al., adult proliferation et 2012a). (Meloty-Kapella and and migration al., the many developing promotes of et stroma in the (Brellier in expressed found tumors is it solid tenascin-C, to Similar family. ein hc a on nafbilrntokta a mostly However, was 1E). this that (Fig. in network fibrils fibronectin fibrillar tenascin-C-positive a the to from in contrast distinct found in was was which trabecular This the region, of 1C,D). matrix Double-label (Fig. fibrillar tenascin-W the region the bulb. in and in overlap whisker extensive tenascin-C and showed gland for the the sebaceous staining surrounding the in immunohistochemistry near capsule and body matrix conical fibrous bulge extracellular outer the the the the in and present of bulge near also the was between was Tenascin-W membrane trabeculae capsule. the immunostaining glassy of matrix the Tenascin-W extracellular in 1B). (Fig. whisker was found tenascin-W mouse restricted of distribution adult the more the contrast, By in 1A). (Fig. widely follicle expressed was Tenascin-C adult the follicle in whisker expressed mouse are tenascin-W and Tenascin-C eut n Discussion and Results ee erpr htbt eacnCadtnsi- r atof part are tenascin-W and tenascin-C both that report we Here, 3 n uhChiquet-Ehrismann Ruth and nvitro in . nvitro in 2,4 hs the Thus, . nvitro in 5111 Journal of Cell Science 5112 ora fCl cec 2 (22) 126 Science Cell of Journal tg faae.OS ue otset.Saebr:50 bars: Scale sheath. growing root 100 actively outer (A–F); the ORS, in anagen. is of bulb stage the Anti- that (I) demonstrates (asterisk). found Ki-67 (arrows), are papilla cells dermal stem the CD34-positive where of Tenascin-W stalk (H) the (arrows). in the papilla immunoreactivity and dermal (C) the capsule to the opening to tenascin-C limited bulb, is the immunoreactivity tenascin-W-rich In a (G) in matrix. embedded extracellular are CD34- cells (F) region. stem trabecular to positive the adjacent in immediately Tenascin-W (E) region bulge. trabecular the the CD34-positive in (D) cells tenascin-C. stem and keratin-15 of patterns non-overlapping distinct, the Double-label showing the (C) immunohistochemistry in (TR). Tenascin-C regions (B) bulge. trabecular the adjacent of niche cell cells stem epidermal stem and proteins tenascin markers. of Colocalization 2. Fig. arwed n nteetaellrmti fthe of matrix extracellular membrane the glassy in the and in (arrowhead) found (arrow). is tenascin-W Fibronectin for (E) positive are as that well fibrils as tenascin-W arrowhead) and (yellow, tenascin-C immunostaining of overlap shows of region regions trabecular Higher the (D) of E. magnification higher and at D shown in region magnification image. the merged indicates The box (C) The (SG). gland sebaceous surrounding the mesenchyme the the membrane, and glassy region the trabecular the bulb, to the restricted near is capsule Tenascin-W near (B) (TR) bulge. region the trabecular glassy and (C), (GM) capsule membrane the to localizes follicles. Tenascin-C whisker (A) mouse adult are in tenascin-W expressed and Tenascin-C 1. Fig. igsns w igus;S hse hf.Scale shaft. whisker 200 S, bars: RS, Ringwulst; sheath; Rw, root sinus; outer ring ORS, sinus; papilla; cavernous dermal CS, DP, asterisk. is found an is with tenascin-W indicated where region The anti-keratin- (K15). with stained 15 is bulge The hair (G) facial follicles. a surrounding in (arrows) found ring is narrow Tenascin-W (F) region. trabecular A h eai-5(1)pstv el fthe of cells (K15)-positive keratin-15 The (A) m (G–I). m m AC;25 (A–C); m m DE;50 (D,E); m m (F,G). m m m Journal of Cell Science a iie oanro igbtentebleadtesebaceous the and bulge the immunoreactivity between ring tenascin-W bulge narrow a whereas keratin-15-positive to limited 2008), the was was al., surrounding Tenascin-C et matrix 1F,G). (Kloepper (Fig. the follicles in hair found facial in expression region. trabecular the the underlying in membrane found was glassy immunostaining fibronectin and tenascin-C both ohtnsi- n eacnWwr on ntefibrous the in found niche. were cell tenascin-C tenascin-W stem both CD34-positive tenascin-W and the 1D), tenascin-C (Fig. of and part Both region are this tenascin-C anti-tenascin-W-stained tenascin-W and in the Because colocalized with largely bulge matrix. the and corresponded region extracellular trabecular closely the of in junction that found the were near cells matrix cluster CD34-positive tenascin-C-positive a contrast, the By near trabeculae. bulge the the epidermal of either of the region labeled cell against CD34 antibody stem and anti-keratin-15 antibodies tenascin-W The or 2D–F). with 2A–C) (Fig. (Fig. labeled keratin-15 follicle, and whisker double tenascin-C the of were cells stem sections of tenascin- populations and different tenascin-C and between W relationships the determine To cells stem extracellular keratin-15-negative the CD34-positive, in surrounding found matrix are 2010). tenascin-W and al., Tenascin-C et (Amoh localized nestin-positive been where have region cells the stem to neural corresponds ring This gland. eacnCadtnsi- lohddsiciepten of patterns distinctive had also tenascin-W and Tenascin-C bevdi te pdra tmcl ihs(..keratin-8- dermis). (e.g. or pads niches not foot in cell was niches stem cell dome immunostaining epidermal keratin-17-positive whisker and the other tenascin-W to in pathway and their observed along Tenascin-C and niche stem bulb. their CD34-positive in of both proliferation and cells to and tenascin-C able migration of be the pattern might influence The they that 2I). suggests (Fig. that that immunoreactivity anagen demonstrate and tenascin-W Anti- in to cells 2H). used was bulb (Fig. was bulb proliferation, CD34-positive papilla the this cell dermal of near the the marker a of membrane Ki67, stalk near the glassy near present the accumulate Tenascin-C was in 2G,H). Fig. tenascin-W found 1C; also (Fig. bulb was the surrounding capsule ihC3-oiiese elahso ofboetnand coated were that fibronectin follicle coverslips or whisker to dishes onto migration, explanted adhesion their were bulges promote cell potentially test stem therefore To on interfere CD34-positive motility. to spreading potential cell the with have cell increased tenascin-W and by tenascin-C with adhesion whether focal accompanied and interfere fibers is stress 2012b) complexes of lack al., resulting The et fibronectin. (Brellier tenascin- and W 2012) Tucker, tenascin-W and and (Chiquet-Ehrismann to tenascin-C Tenascin-C fail on but fibers fibronectin stress on form spread cells stem CD34-positive notecae usrt Fg A.C3-oiiecells CD34-positive 3A). (Fig. substrata coated region bulge the the and from migrated fibronectin onto cells or days, 4–5 tenascin-C, After and tenascin-W. fibronectin fibronectin, with eacnpoen nwikrflils5113 follicles whisker in proteins Tenascin cl as 200 bars: Scale stem cells follicle whisker of proliferation the promotes on Tenascin-C cultured (G) cells fibronectin. than (F) are spread and less (E) complexes adhesion fewer focal have proteins tenascin on cells cultured Stem (E,F) complexes. adhesion focal fibers anti-vinculin-positive stress and fewer have and (D) tenascin-W fibronectin or (C) fibronectin tenascin-C of and mixture a on Stem cultured (C,D) cells fibronectin. on stem spread in cells (green) adhesions focal anti-vinculin- positive and (red) fibers Stress days (B) 5 after explants follicle cells stem vitro follicle Whisker 3. Fig. . nvitro in A tmclsgo rmwhisker from grow cells Stem (A) ausaemeans are Values . m A;20 (A); m m nvitro in nvitro in (B–D). m 6 s.e.m. in . Journal of Cell Science dl 5B6o 2/vmc eecta 12–14 at cut from were follicles macrovibrissal mice mystacial 129/Sv the of or axis C57BL6 long the adult in sections significant Frozen have immunohistochemistry and to Histology Methods 2010) others and Materials al., by et proposed Amoh been value. 2008; neural- therapeutic have potential al., cranial the that of et with population cells (Amoh associated a stem roles of proteins, crest-derived play proliferation Tenascin to and motility. potential migration cell the stem have consistent promoting therefore, are in and cells tenascin-C roles stem of with CD34-positive effects the cultured and on follicles, tenascin-W whisker in cells stem in use or hair differentiation of their populations grafts. to expanding therapeutic prior those cells for tool stem useful follicle-derived a be cells might tenascin-C stem C that neural found of 2010) proliferation al., the culture, et increases in (Yagi colleagues cells of and these niche Yagi of cell and proliferation stem the hematopoietic promotes the marrow, that of bone found part 2012) is al., which et tenascin-C, (Nakamura-Ishizu Nakamura- example, co-workers For and niches. Ishizu the cell stem Under in cells tenascin-C stem G0. studying CD34-positive during of was proliferation cells tenascin-W, vitro the not of the increase but of to nuclei tenascin-C, able phases the here, active described not the conditions during but cells cycle, of cell nuclei the antibody, stains anti-Ki-67 the with which days, stem immunostained 5 and After CD34-positive fixed above. were described of cultures as proliferation explanted the were has the bulges proteins cells, influence tenascin two to the of potential either whether determine cells To stem CD34-positive the of stimulates proliferation tenascin-W, not but Tenascin-C, fibronectin and tenascin-C of mixture a fibers. tenascin-W stress on ( stem few in or cultured complexes relatively adhesion cells and 3C) focal of fewer (Fig. lamellae significantly By mixtures were small tenascin-C There on had 3B). spread either 3D) (Fig. (Fig. that and complexes cells fibronectin CD34-positive anti- adhesion the in contrast, terminate focal that with vinculin-labeled fibers filled were stress and lamellae TRITC-phalloidin-stained broad had fibronectin on migrating 5114 A n a nimueC3 R30;Lf ehoois rn sad NY). Island, Grand Technologies, Life (RM3604; City, CD34 CA; Iowa anti-mouse Bank, Francisco, rat Hybridoma San and Studies IA) (Abcam, monoclonal Developmental rat (TROMA-I; anti-keratin-15 EP1623), (Abcam; anti-keratin-8 monoclonal anti-keratin-17 monoclonal rabbit rabbit (Ehrismann EPR1614Y), 1981), fibronectin anti-human al., polyclonal tenascin- rabbit et anti-mouse 2004), a al., polyclonal et rabbit or (Scherberich 1988), E800 W previously Ekblom, Eclipse and Nikon (Aufderheide described mTn-12 a as with acquired microscope. treated were fluorescence Images Axioscop were 2012b). Zeiss al., sections et (Brellier the immunohistochemistry, ugssta tcudhl ud h tmclspoial othe to sinus, proximally cells ring stem superficial the more bulb. guide help the whisker could the in it to not that but membrane suggests to deep sinus, glassy gradient a cavernous the in 3F). the tenascin-W (Fig. along of distribution promote cells The to tenascin-W bulb. stem whisker potential of the or migration have CD34- the proteins tenascin-C tenascin 3E). ( both either (Fig. spread Therefore, alone less and fibronectin also fibronectin on were cells cultured positive cells in than P rmr nioisue eertat-os eacnCmncoa antibody monoclonal tenascin-C anti-mouse rat were used antibodies Primary , eacnCadtnsi- r soitdwt CD34-positive with associated are tenascin-W and Tenascin-C .5 ramxueo eacnWadfboetn( fibronectin and tenascin-W of mixture a or 0.05) Fg G.Ti scnitn ihterslso others of results the with consistent is This 3G). (Fig. ora fCl cec 2 (22) 126 Science Cell of Journal P , .1 nmxue of mixtures on 0.01) m nvitro in nvitro in n i-re.For air-dried. and m Tenascin- . P , 0.01) in lepr .E,Tee . rnkan . enad,D . ee,W,Faessler, W., Meyer, P., D. Reinhardt, J., Brinckmann, S., Tiede, E., J. Kloepper, himn,R,Ciut .adTre,D C. D. Turner, and M. Chiquet, R., Ehrismann, P. R. Tucker, and G., R. Orend, M., Chiquet-Ehrismann, Heyden, der van J., Ferralli, M., Chiquet, E., Martina, F., Brellier, Heuze M., Degen, E., Martina, F., Brellier, P. Ekblom, and E. Aufderheide, mh . asoa .adHfmn .M. R. Hoffman, and K. Katsuoka, Y., Amoh, otat-abtI Lf ehoois rn sad NY). Island, 568 Grand Fluor Alexa Technologies, and (Life anti-rat Ig goat 488 anti-rabbit Fluor goat Alexa were used antibodies Secondary 97.Aleprmnswr odce he ofv ie,adteaverage the and times, five to three al., of et conducted number (Ostertag the were index by proliferation culture experiments divided each Ki-67 All was from the cells imaged determine 1987). were anti-Ki-67-positive to fields of nuclei Four number DAPI-positive anti-rabbit Ig. total goat anti-rat 568 the goat Fluor and 488 Alexa anti- Fluor were rat antibodies Alexa and secondary and SP6) the clone rabbit and PA; were CD34, Pittsburgh, mouse antibodies were Scientific, Primary cells (Fisher stem above). anti-Ki-67 follicle monoclonal (see whisker immunocytochemistry cultures, for primary of processed proliferation cell measure To assay proliferation Ki-67 10 whisker containing discarded. the were days, solutions cells 5 epithelial After with incubator. contaminated the with cultures to to filled and returned allowed removed, gently and was and then serum with shaft medium was calf previously of fetal dish coated 10% amount The coverslip with small hours. or a 1–2 dish for with plastic containing adhere below) a fragment (see onto whisker matrix placed dissected extracellular was the cells brief, methods stem In published the of 2008). adaptation Hu, an and using cultured (Sieber-Blum were cells stem follicle Whisker and coating substratum culture, immunocytochemistry cell stem follicle Whisker mh . i . asoa .adHfmn .M. R. Hoffman, and K. Katsuoka, L., Li, Y., Amoh, Swiss the References from and support 31003A_135584 numbers acknowledge [grant 310030_125397]. J.C.S. Foundation Science and National J.F. R.C.-E., R.C.-E. Funding and R.P.T. follicle of was sectioning, whisker laboratories tissue work the the in the The jointly out out conducted immunocytochemistry. carried carried and R.P.T. J.F. immunohistochemistry and prepared and experiments, and R.P.T explant experiments the manuscript. conceived the R.C.-E. and J.C.S. R.P.T., contributions Author advice. and and Blankenship Tom assistance Shibata, for Brad FitzGerald thank Paul to like would authors The Acknowledgements a with determined were differences Statistical Student’s calculated. was index proliferation 5 oa dein eepromdo tmclsfloigmtosdescribed methods following cells stem 2012b). on al., et performed (Brellier vinculin-positive were previously of counts adhesions and area, focal cell of measurements Immunocytochemistry, 5 and tenascin-C m eoecluigteepat,patccvrlp rdse eecae with coated were dishes or coverslips plastic explants, the culturing Before eieueu nst akr o ua pteilhi olcese el n their and cells stem follicle hair epithelial human for markers situ niche. in useful define R. Paus, and R. npooigciknmols tahet Mr fibronectin. attachment. native myoblast binds Laboratory chicken Harbor promoting Spring in Cold NY: Harbor, Spring Cold Press. 128-147. pp. Yamada), In modulation. adhesion P. R. solid tenascin-W. Tucker, of human and properties R. most modulating Chiquet-Ehrismann, for C., J. R. tenascin-C Schittny, than Chiquet-Ehrismann, biomarker and cancer C. better Ruiz, tumors. a L., is Terracciano, Tenascin-W Y., Courty, ntemsnhm,siti oeua om,addpnec nepithelial- on dependence and forms, molecular in shift interactions. mesenchyme, mesenchymal the cells stem in pluripotent induced and cells medicine. stem regenerative embryonic for over cells stem pluripotent tmclspooerpi fsia odijr n eoeyo akn function. walking of recovery and injury cord spinal Cycle of repair promote cells stem /lmrn eacnC 5 tenascin-C, murine g/ml 7 x.Dermatol. Exp. 1865-1869. , t M ln Pathol. Clin. BMC ts eulvrac,two-sided). variance, (equal -test m 20) muohntpn ftehmnblergo:teqetto quest the region: bulge human the of Immunophenotyping (2008). /ltnsi- sdsrbdpeiul Bele ta. 2012b). al., et (Brellier previously described as tenascin-W g/ml 17 m /lmrn irnci ln ri obnto with combination in or alone fibronectin murine g/ml .Bo.Chem. Biol. J. 592-609. , xrclua arxBiology Matrix Extracellular .Drao.Sci. Dermatol. J. .Cl Biol. Cell J. 12 m 14. , 18) eacndrn u eeomn:appearance development: gut during Tenascin (1988). /lmrn eacnWo itr f5 of mixture a or tenascin-W murine g/ml n.J il Sci. Biol. J. Int. 107 256 21) eacn n h motneof importance the and Tenascins (2012). 2341-2349. , 4056-4062. , 60 -or’,N,Ptt . rz,T., Kryza, A., Petit, N., ´-Vourc’h, 21) h datgso arfollicle hair of advantages The (2010). 18) oeo cino fibronectin of action of Mode (1981). 131-137. , 5 000gltnbnigfragment gelatin-binding 60,000 20) utptn arfollicle hair Multipotent (2008). 8 e.R .HnsadK M. K. and Hynes O. R. (ed. 187-194. , a 21b.Teadhesion The (2012b). E supplemented MEM (2012a). m g/ml Cell Journal of Cell Science uhisi .N. M. P. Muchlinski, R. Tucker, and R. Chiquet-Ehrismann, M., Degen, V., C. Meloty-Kapella, cebrc,A,Tce,R . aadr,E,BonLei . atn .and D. Martin, M., Brown-Luedi, E., P. Samandari, P., Kleihues, R. and Tucker, P. A., Scherberich, Burger, T., Shibata, B., Volk, B., C. Ostertag, T., Uede, J., Morimoto, K., Okabe, Y., Omatsu, Y., Okuno, A., Nakamura-Ishizu, xrse nkde n tstso oeadsot ucedevelopment. muscle smooth and bone of sites 117 at and kidney in expressed R. Chiquet-Ehrismann, mar bone the Y. in Kubota, regeneration. required and hematopoietic is T. Suda, tenascin-C T., Nagasawa, mammals. other and primates in area foramen vitro. in osteoblasts on tenascin-W of Effects oolnlatbd i6 samre o rlfrtn el nseetci biopsies stereotactic in cells proliferating tumours. for brain marker a of as Ki-67 antibody monoclonal 571-581. , 21) oprtv nlsso irsacutadinfraorbital and count vibrissa of analysis comparative A (2010). caNuohr (Wien) Neurochir. Acta 20) uietnsi-:anvlmmaintenascin mammalian novel a tenascin-W: Murine (2004). Blood 119 5429-5437. , 89 117-121. , o irevrnetpie for primed microenvironment row elTsu Res. Tissue Cell 21) xrclua arxprotein matrix Extracellular (2012). .Hm Evol. Hum. J. 58 334 447-473. , 445-455. , 18) The (1987). .Cl Sci. Cell J. (2008). ai . aaiaa . uui . aaai . rg,T,Kt,K n Yu, and K. Kato, T., Ariga, Y., Nakatani, Y., Suzuki, M., Yanagisawa, H., Yagi, og .E,Prtr,C,DusZmemn,M . oht . iti . Suter, T., Pietri, A., Rochat, T., M. Dours-Zimmermann, C., Paratore, E., C. Wong, E. S. McKay, and P. R. Tucker, P. R. Tucker, ibrBu,M,Gi,M,H,Y .adSee,V. Szeder, and F. Y. Hu, M., Grim, M., Sieber-Blum, Y. Hu, and M. Sieber-Blum, rlfrto fmueebyncnua tmcells. stem neural embryonic mouse of proliferation K. R. rs-eie el ihse elfaue a etae akt utpelnae in lineages multiple to back traced be al. can et skin. D. features adult Meijer, cell the P., stem J. with Thiery, cells S., crest-derived Dufour, R., D. Zimmermann, U., expression tenascin-C n glia. and follicle. hair adult the in cells stem cultures. NCSC) 21) N- ptp-arigtnsi- pie ain euae the regulates variant spliced tenascin-C epitope-carrying HNK-1 (2010). eacnpoen nwikrflils5115 follicles whisker in proteins Tenascin Development 20) bomlnua rs elmig cell crest neural Abnormal (2001). .Cl Biol. Cell J. .Vs Exp. Vis. J. ihmrhln nies oligonucleotides. antisense morpholino with 112 1031-1039. , 175 20) os pdra erlcetse el(EPI- cell stem crest neural epidermal Mouse (2008). 9 19) h xrsino eacnb erlcetcells crest neural by tenascin of expression The (1991). 15. , 1005-1015. , e.Dyn. Dev. 231 ainatrtei iokokonof knockdown vivo in the after ration 258-269. , .Bo.Chem. Biol. J. 20) lrptn erlcrest neural Pluripotent (2004). e.Dyn. Dev. 285 37293-37301. , 20) Neural (2006). 222 115-119. ,