I mmunocyt ochem i cal Demonst rat i on of «- Tubul i n Modi fi cati on dur i ng Axonal Mat urat i on in t he Cerebel l ar Cort ex RI CHARD CUMM I NG, ROBERT D . BURGOYNE, and NI CHOLAS A . LYTTON Labor at or y of Bi ol ogi cal U l t r ast r uct ur e, Nati onal I nst i t ut e f or Medi cal Resear ch, M ill Hill, London NW7 1AA, Engl and . Dr . Cumm i ng' s pr esent addr ess is Amer shamI nt ernat i onal , Amer sham , Bucki nghamshi re, Engl and. ABSTRACT Pr evi ous l i ght m i cr oscopi c i mmunocytochem i cal st udi es usi ng t wo monocl onal ant i bodi es t hat r ecogni se a- t ubul i n (YOL/ 34 and YL1/ 2) but di f f er in t hei r i sot ypi c speci f i ci t y have shown t hat t he unmyel i nated par al l el f i ber axons in t he cer ebel l ar cor t ex ar e l abel ed w ith onl y one of t he ant i bodi es (YOL/ 34) . We nowshowt hat at 10 d post nat al l y t he par al l el f i ber s ar e l abel ed w ith bot h ant i bodi es, and t hat dur i ng devel opment YL1/ 2 ( but not YOL/ 34) i mmunoreact i vi t y di sappear s pr ogr essi vel y f r om par al l el f i ber s in t he l ower r egi ons of t he mol ecul ar l ayer upwar ds t owar ds t he ext er nal ger m i nal l ayer . By - 28 d post nat al l y, t he di f f erent i al st ai ni ng pat t er n of par al l el f i ber s by t he ant i bodi es is est abl i shed t hr oughout t he mol ecul ar l ayer . The ti me cour se, l i ght m i cr oscopi c, and ul t r ast r t act ur al st ai ni ng di st ri but i on cor r esponds to a pr ogr essi ve change in a- t ubul i n i mmunoreact i vi t y as t he par al l el f i ber s f or msynapt i c cont act s . Thi s modi fi cati on of a- t ubul i n (whi ch was not obser ved in Purki nj e cel l dendr i t es or Ber gmann gl i a) may be r el at ed to t he f ormat i on of a basi c i sot ype of a- tubul i n w i t hi n par al l el f i ber axons at mat urat i on . Tubul i n het erogenei t y is most st ri ki ngl y demonst rat ed in br ai n ( 12) . Furt hermore, t he r ange of a- and , B-t ubul i n i sot ypes is expr essed by si ngl e neur ons ( 15) and tubul i n het erogenei t y i ncr eases dur i ng br ai n development ( 14) . The requi rement f or mul ti pl e i sot ypes may r ef l ect t he var i et y of di f f erent f unc- t i ons of m i cr ot ubul es w ithin each neur on dur i ng development and at mat uri t y. The advent of hybri doma t echnol ogy has al l owed mono- cl onal ant i bodi es to be pr oduced t hat have speci f i ci t y f or di f f erent r egi ons of t he tubul i n mol ecul e . We have r ecent l y shown, usi ng l i ght and el ect r on m i cr oscopi c i mmunocyto- chem i st r y ( 11) , t hat t wo monoclonal ant i bodi es (YL1/ 2 and YOL/ 34) have di f f erent speci f i ci t i es f or i sot ypes of a-t ubul i n, and t hat t hese ant i bodi es l abel di f f erent popul at i ons of axons in the cent r al ner vous syst em . To exam i ne whet her t hese di f f er ences ar e evi dent dur i ng axonal mat urat i on, we st udi ed t he unmyel i nated paral l el f i ber axons of t he devel opi ng cer e- bel l ar cor t ex, whi ch have a well def i ned post nat al devel op- ment ( 1- 3) . Our r esul t s suggest t hat i mmunocytochem i cal t echni ques w ith monoclonal ant i bodi es can be used to vi su- al i ze a modi fi cati on of a-t ubul i n in axons t hat occur s dur i ng development . MATERI ALS AND METHODS Ant i body char act er i zat i on was car r i ed out as descr i bed pr evi ousl y ( 9, 11) , usi ng t he SDS gel i mmunobl otti ng t echni que . Cerebel l ar cyt osol was pr epar ed f r om 10- d- ol d and adul t ( 60- d) r at s by homogeni zati on in5 mM Tri s/ 2 mM EGTA, pH 8 . 0, and cent ri f ugat i on at 40, 000 g f or 60 m in at 4°C, and t he prot ei n THE JOURNAL OF CELL BI OLOGY " VOLUME 98 JANUARY 1984 347- 351 a The Rockef el l er Uni versi t y Pr ess - 0021- 9525/ 84/ 0110347105 $1 . 00 concent r at i on of t he cyt osol was det er m i ned . M i cr ot ubul es wer e pr epar ed f r om adul t r at br ai n usi ng t emperat ure-dependent assembl y/di sassembl y ( 6) . Samples were sol ubi l i zed and separ at ed by SDS PAGE on a 6%sl ab gel . The same prot ei n concent r at i on ( 100 ug) was used f or 10 and 60- d cer ebel l ar cyt osol t r acks . Immedi atel y fol l owi ng el ect r ophor esi s, prot ei ns were t r ansf er r ed f r om t he gel ont o ni t rocel l ul ose paper (0 . 45 um ; M i l l i pore Cor p., Bedf ord, MA) by t r ansver se el ect r ophor esi s (in Tri s/ gl yci ne/ met hanol / wat er [ 181) at 10 V f or - - 17 h. Confi rmati on of prot ei n t r ansf er was obt ai ned by st ai ni ng one t r ack of each age w ith am i dobl ack, while t he remai ni ng t r acks were pr ocessed w ith monoclonal ant i bodi es ( asci t es f r act i ons) usi ng t he i mmunoperoxi dase pr oce- dur e w ith di am i nobenzi di ne as subst r at e. Rat monoclonal ant i bodi es YLl /2 and YOL/ 34 were r ai sed agai nst yeast tubul i n by Ki l marti n et al . ( 16) andhave pr evi ousl y been char act er i zed as r ecogni zi ng br ai n a-t ubul i n ( 8- 11) . Mouse monoclonal ant i body Tu 2 .1 was r ai sed as detai l ed by Gozes and Barnst abl e ( 13) agai nst br ai n m i cr ot ubul es and has been shown to speci fi cal l y det ect i sot ypes of 6-t ubul i n. Male W i st ar r at s of 10, 15, 20, 24 and 28 d post nat al l y t oget her w ith adul t s ( 60 d and ol der ) were t r anscar di al l y per f used w ith 4%paraf ormal dehyde, 0. 25% gl ut ar al dehyde in 50 mM cacodyl at e buf f er, pH 7.2 . Fol l owi ng post f i xat i on f or a m i ni mumof 16 h at 4°C, 20 umparasagi t t al sect i ons of t he agar - embedded ver m is were cut on a vi brat ome. Af t er ext ensi ve washi ng in phosphat e- buf f er ed sal i ne, sect i ons were i ncubat ed f or 72 h at 4°C w ith di l uti ons of pri mary monoclonal ant i bodi es and pr ocessed f or l i ght m i cr oscopy usi ng the i ndi rect t echni que (wi t h per oxi dase conj ugat es; M i l es Labor at or i es, I nc ., El khart , IN) wi th 4- chl or al - napht hol as subst r at e . Rats of 10 and 15 d were al so pr ocessed wi th ant i bodi es YL1 /2 and YOL/ 34 f or ul t r ast r uct ur al pre-embeddi ng i mmu- nocyt ochem i st r y, except t hat di am i nobenzi di ne was used as subst r at e ( 11) . RESULTS The i mmunobl otti ng dat a shown in Fi g. 1 demonst rat es t hat ant i bodi es YL1/ 2 and YOL/ 34 sel ecti vel y l abel a-t ubul i n f r om bot h 10 d and adul t cer ebel l ar cyt osol , while ant i body 347