Characterization of isolated mouse cerebellar cell populations in vitro

•laarnaloflVeuroimmunoloSy, I (1981)457-,~70 '

457

Elsevier/North-HollandBiomedicalPress

Characterization of isolated mouse cerebellar cell populations in vitro Jutta Schnitzer ~md Melitta Schachner * Del~zrtmem o1"NeurobioloD,, Universily of Heidelb~r& Ira Neuenheimer Feld 504, 6900 Heidelberg (F.K G.)

(B.ece~ved16April, 1981) (Accepted19May, 1981)

Smm.ry Cells from early postnatal mouse cerebellar cortex were isolated by discontinuous BSA gradient centrifugation. Three cellular fractions were obtained and called A r(int¢fface at 0-10~ BSA), B C.0-15%) and C (15-25%) these fractions were characterized after maintenance in vitro for 3 day§ by indirc immunofluoreseence labeling with several cell type-specific probes: Terminustoxin was used as a neuronal marker. Under the described culture ~onditions "Ihy-l.2 antibodies served as additional marker.~ for mature neurons and NS-4 antisermn for neurons and oligodendrolllial fells. Glial fibfillary acidic (GFA) protein was used as a marker for differentiated astroglia, and fibronca::tin as a marker for fibroblasts. Monoclonal antibodk~ to 04 antigen and antise:am to corpus callosum served to distinguish

eltlede~kotl~ Fraction A contaim most of the cellular debris and cells with large cell bodi~ (about 20/tin in diameter) which ate positive for Thy-1, NS-4, and tetanus toxin. 1~3" birthdate labeling with [3H]thymidine these cells can be identified as Purkinje cells a n d / o r Goilli type II M s . Fract/on B is relatively heterogeneous. R contains predominantly GFA protein-positive astroglial c~.'lls (about 50g of all cells) which can be clamfied into 3 morphologically distinct cell ,:ypes, flat epithelioid cells aml mtr4haped ~ with .~ "ck or very thin cellular processes. Fraction B is enrichect also in 04 antilten.positive oligodendrocytes, fibronectin-positive fibroblasts and Thy-i negative, but NS.4 and tetanus toxin positive c~lls with small cell bodies and many free processes. These small neurons, putative stellate and basket cells, have many free procesr~ and ~xe morphologica~qy different from the bipolar putative This v~rk ~a~msupport~ by Mtsche Forschungsgemeinschaft.J. Schnit:,.eris a granteeof St~d~cstittu~ des Mughm Yolked. * To wllomootrmpoadea~g~ukl be sent, 0165-5728/81/0000-0000/$02.75~ Elsevier/NotCh.HollandBiom.'~calPros

428 granule cells, some of which are also present in this fraction, Fraction C contaifis predoj~la/r.antly smal~ neurons, mostly patative granule cells (mote than 90~ of all cells) which are posidve for NS-4 and tetanus toxin, but ta~vative for Thy-1.

Introdm'tion To understated the molecular mechanisrl~s of cell interactk3n$ in the developing and adult nervous system, ~ populatiom of cell types aml subsets will be needed to .~tudy their behavior in vitro, The advantages of the rmyase cerebellar eantex for such studie~ have been indicated in the companion paper (S¢imitzer and Schachner 1981a). The aim of the present study was to further develop a previously descrilw.d cell separation method (Campbell et al. 1977) for early postnatal mouse cerebellm" coxtex and to account for each cell type ~ing establidu~ markers in indirect inv munofluorescence procedures (Schadhaer and Willinger 1979; SchnJtzer and Schaclmer 1981a) Furthermore, to define sub~'~s of neurons, t4rtlulate-Labeling methods were emplo).ed. Ma~flds and Meflmds

Mice C57BL/6J mice were maintained in our breeding colony at the deparlment.

Preparation v/the cerebellar cortex Cerebella of 6-day-old C57BL/6J mice were cut f~ittally into 6 pieces, each approximately I mm thick. The u~ctions were placed ut~kq"a diHcclion mk'fcw,fope. with the cut fide face up. The rel~on of th~ deep c . . e ~ nl~c:l¢iwa~ l h ~ with fine scalpels. After this procedure the, cerebe|ia~ folia could be anfolded into a sh¢~et-like ,¢tructure.

Preparalion o/ single cell ~,~¢pensionJfrom early p~matal mouw ct~'¢b~ and cell culture Preparation of single cell suspensions frtnn early postnatal mome caebdltm, and maintenance of monolayer cullures were carried out as d ~ r i b a l previou~ (Schnitzer and Schachner 1981a).

BSA step gradient isolation of cerebcllar cortical cells Bovine serum albumin (BSA, Path.O-Cyte Vo 35% slCril¢ sOlUtiOn)was purchm~ front Miles (Miles Laboratories, lnc., U.S.A.). A 3~step BSA gradient was prepared ustag a modification of a previot~ly desc6bed procedure { C m p b d et al. 1977). The BSA stock solution was diluted with I ~ a l Medium Eagle~s (lIME) ~ M ~ I ~ Hanks' salts to the followin8 concentratio~ 2~$%, 15%, and 10%. Cdlulo~ nilmle

459

ceatrifu~;,e tubes (1.27 × 5.08 ram) were filled with ! ml of each of the ~SA solmio~ starting ~ith the most concentrated one. A single cell suspension of freshly d h ~ t ed 6-day-old C57BL/6J mou~ cerebellar cortex (8 X 10°/0.8 ml in BME) w;~ the~ loaded en top of the step gradient and centrifuged at 10,000 X g and 14°C for ! h using a Be~kman rotor (SW 50.1) in a Kontron TGA 50 ultracentrifuge. Ceil~ were collected at the 3 interfaces with a Pasteur pipette. Cells of the BME-10~: BSA interface were collected in fra,:tion A, from the 10~-155 interface ia fraction B, and from the 155-25~ interface in fraction C. Materia! which collected at the botu)m of the cenuifuge tube was mostly either dead cells or erythrocytes. Each fra::tion was diluted with 3.0 ml BME and centrifuged for 10 rain at 4°C and 64 × g. The upl~r two.thirds of the supernatants were then discarded, the remaining p(xtion diluted once more with 1.5 ml BME and centrifuged agai,1 for I0 rain at 4°C and 64 × & The supernatant wa.~ then completely and carefully removed. Cells w~e then resuspended in sterile culture medium, consisting of BME with E~trle's sa~, 105 hor~e sentm, 2.5 mg/ml glucose, 2 mM L-glutamine, 100 U / m l penicillin and 100 itg/ml streptomycin at a concentration of 1.5-1.75 × 106 cells/ml. Dead cells were determined by trypan blue dye exclusion. 50 pl of this cell suspension were plated onto poly-L-lysine coated glass coversllps (0.6 cm diam.) and maintained in a humidifie~ CO2 incubator supplemented with 2.2T CO2 at 35.5bC, as described (Schnitzer and Schachner 1981a).

Antibodies and tetanus toxin Antibodies to NS-4 antigen, GFA protein, Thy-l.2, fibrouectin, 04 antillm| and tetanus toxin were used as described previously (Schnitzer and Schachner 1981a). Antiserum to corpus callosum has been described by Schachner et al. (1977) ~md was used at 1:30. At this concentration the particular batch of antiserum was gu3~sn to react delectably only with oligodendrocytes, and not with astrocytes. Antibodies to bovine GFA protein were obtained from Dr. L.F. En8, to tetanus toxin from Dr. V.IL Zurawsky, to Thy-i.2 from Dr. G. Hlmmerling, and to fibronectin from Dr. P.O. Hynes. Tetanus toxin was obtained from Dr. E. Haber,'naon. Reagents fc~" indirect immunofluorescence procedures have been de~'cribed previously (Schnltz~ and Sdutchner 1981a). lndirea lmmunofluorescence procedures Indirect immunofluoresceace on live ~nd fixed monolayer cultures was carried out as deseribed previously (3chnitzer and Schachner 1981a). •Birg~dale labeling by [JH]thymidine lhre~mmt NMRI females were injected intraveneom,l'- through the retroorbitai pimm ~ith 400 ~ [3Hlthymidine (29 Ci/mmole, 1 mCi/ml, ~3m Amm'sham Bin:bier, Frankfurt, F.ILG.) at days 11, 12 and 13 of gestational age when Purkinje and Oollly type II cells are born (Miale and Sidman 1961). Day0 of embryonic age w u ¢lk~plated as the day a vaginal plug was found. The dose of radioactivity was 10 FCJ/$ w=isht. Cetebellar cortices from 6-day-ol~L fittermates ~ ' e prepared and dilm¢isted into single cells as described above. Single cell sus~nsio~ wece m~xl to

460 make slide preparations with a Shandon Elliott cytocentrifuge (Campbell et al. 1977)..Cells resuspended in 0.5 ml pin.pray.e-buffered saline, pH 7.3, containing 2~ horse serum (PBS.HS) were immediately treated with 0.5 rnl 4~ l~raformaldehyde and 0.5~ glutaraldehyde in phob-phate-beffered saline, pH 7.3 (PES), for 15 rain at room temperature. CO~ ,.ere diluted wi~h PBS-HS to a ~ t r a t i o n of 3 × 10~ cells/ml, Of this suspension 0,7 ml was applied to a well in the 6.Titocentrifuge and centrifuged for 5 rain at 600 X g. Slides containing cells were i~mersed into 4~ paraformaldehyde in PBS for 15 rain at room ~atperature, washed 3 times with PBS (5 min each, at room temperature) and finally 3 times with distilled water. They were then dried at room temperature in a dedicator, dipped in photographic emulsion NTB-2 (Kodak, diluted I : I with distilled water} and exposed in tk~ dark at 4°C for approximately 3 months. Slides w~re developed with Amidol (0.023 M Amidol, 0.143 M sodium sulfite, (~,0067M potasfium bromide lull obtained from Merck, F.R.G.] diluted in water) f-3~20 min at 14°C, fi;ted, washed with d~stilled wa~r, and dried at room temperature. They were examined with a Z¢iss IM 35 microscope with Nomarski opdcs, Ceils wi:h more than 10 grains over the nucleus were c,ou~ted as positive.

Results

Fractwn A

This fraction collected at the interface of 0-10% BSA contains large cell bodies (approximately 20 #m in diameter), some small cells, and a considerable amotmt of debris (Fig. in). Approximate|y IS of all cells of the unseparated total cell population are recovered in fraction A (Table I), About 65~ of the small cells in this fraction are dead as judged by uptake of trypan blue. They start to di$integrate after a few hours and remain as cellular debri~ in culture. The remaining cells settle in culture, but rarely extend processes and do not even ~cem to attach properly to the poly-L-lysine coated 81as,~eove~,slip. The lars¢ cells exprcr~ Thyol ,ntil~l (Fig. lb) already after 2--3 h in vitro, NS-4 antigen and tetanus toxin receptors on their surfaces (Table 2). After several days in vitro these l a r ~ cells start to di~inlelffate. Small dead cells and debris are hi[hly0 but mmobpecifically fltwrt,,~,,t'n! with ally antibody even when first antibo:ly is omitted and only ur.oml flttore~r.em antibody is applied. Large cell bodies in fraction A are n ~ stained by antibodm to GffA protein, 04 or corpus callosum antigens nor fibronectin. To decide when these neurons are born, birtMale.labelin$ experinmttls wi'~h [3Hlthymidine were performed at embryonic ,~ys ! !, 12, and 13 when Purkinje ned Golgi type Ii cells are born (Miale and Sidwan 1961). At postnatal day6 pulse-labeled mice were killed, cerehe!!ar cortical celh~ u~mated by ~ A gradient centrifugation and incorporation of l J H i a h ~ a i n e measured by auu~r~diol~apby of slide preparations of separated cell fractions. Figs. 2b-e f,how ceili of f ~ A . Some but not a!l large cell bo~ies carry ~lutoradic~aphi¢ ~p'ahut ¢~'er the nueletui. Autoradiography of ceil preparations from fmctio~ B and C did not show any labeied ,:ells (not shown).

Fig. I. ~ l l n g of Thy-!.2 (b) in fraction A in 3-day-old cultures of 6-day-old C$7BL/61 mouse cet-cbellum.A cell with a large cell body (~,bout 15 ~tm in diameter) and no processes (large arrow) shows ring fluorescem~ staining with anti.Thy-l.2. This fraction contains also cellular debris (sm~ll ~ ) which is non,specifically fluorescent, (a) Phase contrast micrograph. Bar denotes 20 ~tm. T h e enrichment of large cell bodies (Purkinje a n d / o r Golgi type II cells) i n fraction A is shown in Figs. 2a (unseparated cells) a n d 2 b - e (fraction A). Large a n d small cell bodies in Figs. 2 b - e are larger compared to Fig. 2a. This swelling m a y be due to BSA gradient centrifug~tion procedures. TABLE I CELL YIELD AND VIABILITY IN FRACTIONS A, B, AND C OF DISCONTINUOUS BSA GRADIF.NI C..¢llsfrom o.rebellar cortices of O-day-oldC57BL/~J mice were counted and scored for viab~ty af).~ ,.olkct/on from the gradient interfaces. Fnmtion

A

Cell yield of total numberof cell~,loaded onto gradient 0.9~

B

4.~,'S

C

50.0~

Cell viability as judged by exclusion of trypan blue 67"+"3 ~ a 75±5~ 90_ + I

• M~nly mall cell bodie~in fraction A take up tryp~, blue, while only few cells with large cell bo~¢:~ate Uypm blue positive.

462

Fig. 2. Birthdate determinations of large ncmm~s in f~a~/on A by | ~ H ] ~ i ~ . m p m a t ~ Pub¢ labeling with [3H]thymidin¢ wa~ perf,-~rmed at ~,~ab:cy¢~/l/¢day 12. i~/$,a ~ w s a alJd¢ In't'lpafa~ of

463 'FABLE 2 IMMUNOCYTOLOGICAL CI~,RACTERIZATION OF CELL TYPES IN FRACTION A, B, AND C OF D'~SCONTINUOUS BSA GRADIENT Cells from cerebellar cortices of 6-day ~l,t C57BL/6J mice were collected from the BSA step gradient and maintained on poly-r-lysine coated glass coversliFs for 3 d:~ys in vitro as described under Material and Methods. Cells were then processed for indirect immunofluorescence on live or prefixed cells depending on the antigen. Fra:fion

Cell type

Identified by markc~

A

Purkinje and/or Golgi type II

Thy-i and NS-4 antigens, tetanus to,,in receptors

B

Astrocytes Oligodendrocytes

GFA Frotein 04 and corpus eallosum antigens

50 ± 10 (6 ± 2)

Fibronectia NS-4 anti~,en and tetanus toxin receptors

5 _+ i ( 1.0 -~0.5)

Fibroblastic cells Small neurons (putative basket, stellate, and granule cells) C

Small neurons (mostly putative granule cells, few basket and stellate cells)

~ Immunolabeled cells (numbers in bracl~ets represent in unseparated cells)

90-4-5 (< 1)

6 ± 2 (2.5 +0.5)

39 ± 18 (90 -4- I)

NS-4 antigen and tetanus toxin receptors

96 _ 2 (90 ± I)

Fraction B This fraction contains cells ~oilecting at the interface of 10-15% BSA. It consists of 4 ~ of all cells applied to the gradient (Table 1). A b o u t one fourth of these cells are dead as j u d g e d by uptake of trypan blue (Table i). W h e n these cells are maintained in culture for 3 days and immunolabeled for identification of cell x y ~ s , a 9 - 1 0 - f o l d enrichment of G F A protein-positb/e astrocytes (Fig.3) over nonseparated cells is seen (Table 2). These astrocytes appear in three morphologically distinct cell types, flat epithelioid cells and star-shaped cells with thick or very thin cellular processes. Oligodendrocytes as identified by immunolabeling with antibodies to 04 and corpus callosum antigens (Fig. 4) are enriched by a factor of 2 - 3 (Table 2). unseparated ~ g l e cell suspensions of postnatal day 6 cerebellum to show the degree of enrichment of large ~ in fraction A (Figs. b, c and d, e ). Figs. b, c and d, e show slide preparations of fraction A of 6-day-old cerebellar cells. Some large cell bodies (large arrows) but not others ~small arcows) carry autotadiographic grains over their nuclens. Cells with small cell bodies carry no autoradiographic grains over their nucleus (b, c and d, e; arrowheads). Note that large and small cell bodies i~ Figs. b, c and d, e are larger compared to Fig. a, possibly due to the different cell preparation 9rocesses. Figs. b, c and d, • represent the same visual field, respectively; however, interference contrast settings are different in e and • from those in b anO d. Bar denotes 20/~m.

464

Ii~,. ) lmm.~.,qab¢lin~ (,~"¢;FA pr(~tt:in t h) in flr;r~tit~nB in .~.~;~,.-otdt:~ll~r¢,'.~,Ar6.daz~-~ C.~71tL/61 v,ifh ,.'p~d~,..I,,~d~)l,,rptl~,h~g; " | ~ t'cll~,','. th thin pr,~,*'~,~. ~

~t;~r-~.h,~ t ~ l p J ~ ,

d;~'r~'~,) arc ~ !

tJcn, )1£', 2(1 !I Ill

i:ihrohiasls or fibre)bit.st-like cells cxpres,,il~g fihr~n¢¢tin ;Ire enriched h~,' a f~ltor of ;lppr~xinl;flely5 ('1;m1¢2). "lh¢ t~c~urrcn¢¢ t)f ncur,~ns wilh ~Jqrmll eel! I'x~lit.'~ is redttcett 2 3-fold over the hi)n-separated pepul~,l/on o | ¢¢rch~,J|ar cortical cells. T h e Edentit~: of these ceils ;ts neurons is confir:~¢~,! by cxprcs.~i~)r, o r ~ 4 a n t i ~ l i and '..etan ,,; t,.,xin recept,)r~ (Fig. 5) and the lack ~f (~I . m i ~ e ~ . il i~, in~er~ting to hole th;,t ,n;,ny of the'~e neuron*, ~ i t h small cell !~odies are star-shaped in a p i ~ a r a n c e and show ;~ differ:~:t morphol¢)gy from olher ~ma|| neurons '~.'hich are ~)il:~lar and which ;ire readily seen it, sparsely seede~ cells in cuJlurc.s Of fraclion C. These sta~r-sh~l~! m.'~=r,,n, arc mor,.- frequently seen in fraction B [ban in nc~-scparazed cerel~llar

~5

Fig. 4. lmmunolabelingof "corpus callosum" antigen (b) in fraction B in 3-day-oldcultures of 6-day-old C57BL/6J mouse cerebellum.This figure represents a selected field of cells obtained in fraction B after 3 days in vitro. Oligodendrocyteswith an elaborate network of processes are stained by anti-"corpus callo~it,m" antiserum (b; large arrows). Another cell with smaller cell body and less elaborated processes (a; small affow) is negative(b; small arrow). (a) Phase contrast micr~graph. Bar denotes 20 ~m.

cortical cells. Thy-i positive neurons with large cell bodies are extremely rare in fraction B. None of the small tetanus toxin-positive neurons are labeled by Thy-I antibodies (not shown).

Fraction C This fraction contains a.ll cells collecting at the interface of 15-255[ BSA. It contains about 50'$ of all cells applied to the grad:ier~t (Table 1). Approximately 905[ of these cells are intact as judged by the exclusioa of trypan blue (Table 1). Whe~ lhese cells ate maintained in culture for 3 days and subsequently immunolabt:led for identification of cell type s approximately 955[ of all cells are labeled by tetanus toxin (Fig. 6) and antiserum to NS-4 antigen (Table 2). Less than 1% of all cells in fraction C are oligodendrocytes or fibroblast-like cells. Some astrocy~es arq.- present in this fraction. Their shape is more uniform than of astrocytes in fraction B (Fig. 7).

l:ig. 5. lmmunolabcling of t e t ; m u s f~,xin re-.cCp~, in fracfh0n B in 3-day-rP, d ~Jtuf¢~ of 6-day-oW ('57BL/6J mouse cerebellum. Thi~ figqr¢ repr¢~nt~, a ~¢Icc~ ~i¢td ~howin$ n~ny t¢~'l~ ~xin.posi~iv¢ cells with relatively .,,mall cell b~lien an~ mu|tipL~ pro~:c~'s ~(J;~g¢ arro~,'~,). Ar~ ¢j~thg~i~i~Jcell pr."~babl~y a n astrocyt¢ ( a: .',mall arrow), is not stained (h; ~mall arrow), g# P ' ~ ~ ¢ r a > l migrograph. Bar donors 20 I~ m

Fig. 6, Immun.labeling of tetanus to~in reeepto¢~ in f r a c t ~ C in 3-daly-okl ¢ ~ l ~ ¢ ~ ~ 6-diL,-o~ C57Bl./6J mou.,,c cerebellum, This figure repre:~cnts a fief! c.f ee|b ~ylpi¢~ for frace~rJ C. C¢~s ~'ieh ~ r ~ i cell bodie,,, are )clans:, toxin-positive { h). (u)Phg..~¢ c(~ntr,,~,t microgf•ph. D ~ deno~e~ ~ ~n~.

467

Fig. 7. lmmunolabeling of GFA protein in fraction C in 3-day-old cultures of 6-day-old C57BL/6J mouse cerebellum. GFA protein-positive astroglial cells (arrows) are present underneath neurons ,~,ith small cell bodies. Astrocytes in fraction C are generally not epithelioid in morphology and often carry several slender processes. (a) Pl'oasecontrast micrograph. Bar denotes 20/.t m.

They have small cell bodies extending usually between 3 and 5 relatively slender processes. Large neurons are absent ir.~fraction C.

Discussion The present study has confirmed the ust*ft~lness of bulk isolation methods using mainly buoyant density as a parameter to ,:nrich identifiable cell types from early postnatal mouse cerebellar cortex. The method used has the advantage of speed over alternative methods, such as fluorescence activated cell sorting (Caznpbell et al. 1977), which can be problematic when minor cell populations are to be enriched. In comparison to isolation at unit gravity (Rose 1967; Barkley eta]. 1973; Cohen et at. 1978) our present method represents a saving in time of about a factor of 2. However, in contrast to an isolation by fluorescence activated cell sorting homogeneous cell populations h;tve so far not been obtained. A combination of the two methodologies, first bulk isolation then followed by fluorescent activated cell sorting, seems therefore advisable, to assure a quick bulk er,~fichme:at of cerebellar cell types and a subsequent selection of particular celi populations by means of a suitable cell surface marker. The present investigation was undertaken to recognize most, if not all cell

6.68 popu~ions of early pounatal mouse cerebellum~ euablht~ immeaoo~lo~cal markers a~d to quantifycellyields,percmuq~e of ,leadcell,and viabilityof the separated ~lls i~ culture,Another aim of this study was to ~blith a procure W eliminatecelltypes ~ do J~otb e k ~ to the cerebdlK ~ iUt~If, such as neurons of the deep c~rdz~u" n u ~ whkh are p f ~ t ia conventional cerebellar~I~ l~repatations,but have so far been ipofed in the duua~erizatioa of cerebe~r celltypes. Our reudts show that the total cell yield af ~r a disc~atinee~ BSA Uep sradi~t procedure amounts to about half of the input, Cellular debris and wm~ dead cells are colle~.'ted from fraction A, which comtitme~ the eppamo~ step of the dJr~ontinm~us j,,rad~,nt. It is not known how and where about haft of the ~ cells disappe~-. However, a loss of 50~ is not ~ in euiaipalatiem "of freshly trypsialzed and ~ y dissociated cells of early ~ mouse c e r ~ , wh/ch have not been allowed to recover fro~ the ~ t i o n procederes (Campb~ et al. 1977). Vi,~ility of cells as judged by ~ blue dye egehtsitm ranses from approximately 65~ in fraction A, which ¢onlaim mo~ ¢¢llelat d e b ~ and dead odb wi~ small "bodies, w apptoximatdy 9D~ ~ in frzetinn C. The ~ dead ~ in fraction A disintegrate completely within 3 day~ of ¢ultufe such that the majority of surviving cells (appro "ximately 90~) ¢43~U~ of neoroas with ~ cell bodies. Identificatk~n of cell types in the 3 ~epatated frac',h~s was attaapted by means of p:'eviouslyestabEshe~ matker~, For fraction A, an ~litioaal criterioa had to be evaluated to aw,ertain the ~erviving ceils t~th large cellbodies as Pmkinje and/or Golgi type II neurons: birff~date ]abei~ng using a pult~ of [~H~h~midiae and sub..~luent autoradiogtaphy of hola~d cetb. Since latle neusoms of the deep cerebellar nuclei ~,hich ate born at the uaue time as the latBe neurons of the cerebellar cort2x (Miale and Sidman 1961) a~e depleted in oer ~ p~eparatio~, the identification of these NS-~, tetanus toxin-, and 'rhy-l-positive neerons now to be more fi~inly e~tablished than by e~ll size and purely mmphoklli~ criteria which are of ~imited value in d i ~ i a t e d cells and ~ y c r eulmt~ A d / ~ between Purkinje ~nd Golf,i type ii neurons has proven i ~ i b k by th~ presently available. Fraction B is the most heterogeneous ha ¢¢11 type of i~l flr~l~lll, It contaim enrichmer, ts of 9- lO-fo14 in astioeytes, 5-fold in fibrob,lasts of fibrob~.like oelk and a dep'etion in small neurons of about 2--3-fold. It ~ not.,q~tthy that these u u l l net~rons show 2 distinct raotpholos~, one with bipolar app¢~aace of ptoc~tm and anether with ~veral fine p r ~ extending from the c¢11bodies. Whetha these two types of mmrons represent Igranul¢ eeib on one hand of ~.eilate and basket cetb on the other reraains at present elu~ve by i ~ a t m o l a ~ a l crimia llig~ affinity GABA uptake ~ been tak~m as • ~dtetiea for ideatififatioa of stellate and basket cells in cerebdlat cultures ( ~ 1974; ~ 1977). ~ also GFA protein-positive astrocytes show hillh'~ffmity uptal~ in ~ cultur~ (Sclmitz~r and S~ha~hn~, unpublished re~ll~..;) and some astro~'yt~ bare • ~Ur-shap~ pearan~ with many fine cellultrp n g e r ~ , OABA upt~_.ke alone may ~at be sufficient to unequivocally identify stella:e a~gl ~ cell& Attempt, to a b o i ~ Illial uptake of GABA by/~.alanine (Ctm'ic and Dutton 1980) ~ ao~ ~ f u l in our

h~nds (Schnitzer and Schachner, unpublished resuh). Combined autoradiography with [3I-I]GAY3A a~d immunofluorescence for cell ti',,pe specific markers has failed, since GABA is lost continuously from cells during i~'amunofluorescence procedures :ffter .mild frgafion with 81utaraldehyde. Glutaraki,~:hyde concentrations used to retJtin most of intracellular GABA are to,~ high in i,,~munofluorescence procedures sir~ce they increase fluorescence backsround levels s~,: h ~hat specific immunofluorescence is no longer recognized. Thus, other possibiliti¢~.~, will have to be searched for to distinguish stellate and basket cells from gr~nule ceI l~. Fraction C contains the majority of small neurc~:!s .rod most o f these seem to belong to the group of small bipolar neurons. We car,:~o~ exclude, however, that few basket and stellate cells are present in fraction C, ~i:~ce morphological differences between small cerebellar neurons might be the resutl of different cellular environmenU; in fractions B and C and not due to distincth:,ns in intrinsic morphogenefic

dispositions. The enrichment in these small neurons is small, si~t,:~they constitute only 96~ of all cells in this fraction compared to 90~ :in ~ l : , : w a t e d cells. It is not known whether neurons with small cell bodies in fraction ~.: are distinct from those in fractionB by other criteria than morphology of c:!:lular processes, such as for instance birthdate or stage of cell cycle.

Aelmov,l,~lMmmm The authc~xs a~'e grateful to Dr. E. H a b e 1 ~ n n for >urified tetanus toxin, to Dr. O. ~ l i n g for monoclonal antibody to Thy-l.~, to Dr. V.R. Zurawsky for antiserum to tetanus toxin, to Dr. L.F. En[~ for anti~ m m to G F A protekt, and to Dr. R.O. Hynes for a n t i . r u m to fibronectin. We, thar~ Dr. Spring for the use of his m/eroscope with Nomt.rski optics.

Itdm [ h l ~ , D.S., L.L. Italdc, LK. Chaffee and D,L. Wong, Cell ,sep~ration by ve!ocity sedimentation of postm~tl mou~ caebelltmt, J. Cell. Pl~ysiol.,81 0973) 271-280 C ~ G.LoM., M, $chschnt,r atld S.O, Sharrow, Isolation ,:f glialcell-enriched and -depleted popt,l s ~

from mouse ~rcbcllum by dmsity grad/cntcentrJ~uga~on aml electron/ccell~ ' ~

C ~ J., P.. ~ a ~ • ~k,t~o~ml ~ ,

F. Hsjm, D.N. Currk and O.R. Dutton, Separation of cell types from the ~ Ibis.,148 (1978)313-331.

C1ar~ D.N. md G.P,. Datto~ [3HJGABA uptsk¢ as a marker J )r cell type in p ~ u r y culottes of t,~nJbcdhll ~ olfactory bulb, Brain Res., 199 (1980) 473-481. LinkS, ~ ~ uptake of [3HIGABA and differentiation of stell~ c neurons in cu,~ures of dissociated pmms~l rat ~ , m , Bran R~s., 69 (1974) 235-2M. Mgm~r,A., The miMtemm~ and kk~tification of mouse cerebell~ granule cellsm monolayer cu~.ture, P.m., DO (tg"r0 I- t2. Mislr., !~ Slid ILL, Sidsmm,An autoradio~tphic analysis of histogt',tesis in the mouse cerebellu~ Exp. Nawat., 4 (1961) 277-296.

470

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