cGMP accumulation by spinal cord neurons during the period of neuromuscular junction formation

.Veuro~;ence Letters, 30 (1982) 263-;.68 ?.tsevier,:North-Holland Scientific P,~biishers Ltd.

263

¢'GMP ACCUMULATION BY S P I N A L C O R D N E U R O N S DUR;.NG T H E PERIOD OF NEUROML:SCULAR JUNCTION FORMATION

CHERYL L. W E I L L

Departt:)ents of Neurology and Anatomy, Lotasiana Stale University Medical Cente:, P,,,w Grleans, L,,4. 701i2 (ILS A.) (R~ei'~ed October 2rid, 1981; Revised version received March l lt~, t982; Accepted ~:arch 17"h, 1982)

A 4-5-fold inch:use i.~ cGMP levels in spinal cord neur.)ns has bccl~ demenstrated it: vitro and m vi~o upon as.' ociation v, ith sl,-eletal muscle. The increase in cG:",~P was temporally coincident ~.'.ith the on,or of neuromuscular junctior, formation and iadq.endent of any co:~cum',tant change in cAIViP. .rh." respo~;~e ,~as spcdl'ic for spinal ,:ord neurons cocul,.'~:ed with s'.el::ial muscl.:; ,,lie.i ~:dl:~ct')tt!d dot be su,~,~it,,~ted :"or spinal cord neuron; nor ceuld fibroblast'~ be substituted for ,nusdc celk. -lh.. r:spo):~,e x~a, nu) dependent up,on action potential acti,,ity, s:naptic transmis'don or mu_-,;:lc contrac,.ile activity. Mu.~cte conditioned medium w~.s found to produce ~he largest incre~'.se o f .-(.iNIP in spinal cord neuron,, ;~f 23;#old.

To date the molecular comn-)~mication mecb.ani.~ms ~hat :,).tb~crve ';ynapt','..~,.')v.:'u,~ ,,,';:,~i~iz functions and associated structures. Compelling supt;url .~)) rcq,,,ir.:d inIr:cd~,da~ c o m m u n i c a t i o n comes from the observali~.)a d~-..', mo~oncurozv, wit3 di.~ it th:::,' do ::ot m a k e contact with muscle [13]. In addition, several examp!e~; o" ~iv: reguh,.ti,.)n .)f the cellular" f u n c t i o n of either he,roy, or targ,:t c.,~o " ' " by a secre.'.~d f~.cv.:-r of the counter cell have been described [7, 12]. Elucid,',.tion ~t t!:e n~echa~)~_srns by ",,,hick) tissue factors m e d i a t e the regulation o f unique cellular fu~:cfions in specific pa-~ner ,:ells is essen:ial to an ,anderstartding o f n e r v e - ~ u . s c l e s3,naptogene~is. 1 report he, ¢. or) the accumui~tion o f guv..nosir)e 3' : 5'-tnono~hc)s.~hate (cCJ i:v~:,~ •~y chick spina! cord neuron~ without a concor:'.,itam c).a~.tage h~ ader:~.;:,ic~c 3' :_5'm o n o p h o s / , h a t e ( c A M P ) during.; neurontuscular jvr;ction , o r m a t i o ~ in vi ro :-.',:,d ira •dvo. Ceed;J:ioned rnedhtm e x ~ : ' i m e n ) s s).~.~g;.~) th~:t this incrca.-;e i~z cCi?,,,!P c~..~>',2c e:;.~ecte,J O,,a m~:sct,~ factor 'Yo it6 ]-:;~,','/!';'.ig'~ -'.)i~.,;: :~ ,; ?i:;: ..~:;r;~ t :,:) -2:::.t~.:: ~:,.~ a s u s t a i n ~ incrtase in n~'ttrenal cGt, d F levels r.:s a conqequev,:e of (.;~rgct a~::<,c::~57~:, or contact i-~de)~'end,,.~;~,of syn~..plic ac~iv~y. 0304-3940/'82/(~C.¢}--(~)r;~)/$ G2.75 ~> 19~2 hi~.~ic~/.',;<.,,d,-V.'/,i.::r,:, .:::.'.;;'.!u.': v'.:~i~'.;,~.;: ~..i~.

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cAMP and c G ~ P levels were determined using commercial radioimmunoassay kit~ obtained frorn Nev, England Nuclear, Boston, MA. Emb~'yonate~ White Leghorn eggs ~e-e obtained from Truslow Fa'ms, Chestertown, hiD, and Awarenes.,, Fad-ms, Pearl River, LA. Culture medium, horse serum, and culture reagents w'ere obtained .fi'om GtBCO, Grand Island, NY. Embryonic tissue was dissected from appropriately staged embryos according to the staging series of Hamburge:" and Hamilton [6]. A~I culturc~ were prepared in 35 mm collagen-coated Falcon tissue culture dishes and .maintained in a huntidified atmosphere of 5~0 CO2-95°70 air at 37°C. Cells were plated and m,tintained in Eagle's minimum essential medium (MEM) supplemented with heat-inactivated horse serum (100/0 by volume), chick embryo extract '.5°"0 by volu,ne), !:lutamine (2 raM), penicillin (50 U/ml), and streptomycin (50 .,v.g/ml). l~..u+cle, spinal cord aqd muscle-spinal cord cocultures were prepared as previously described [16]. MuscIe cultures were treated with I0 -s M cytosine arabino:~idc CaraC) for 48 h when fusion was 80-90~0 complete. Spinal cord at.,d cerebel!ar cells were plated and cultured in the prec,ence of 10- 5 M araC for the first 48 h. Muscle-spired cord and muscle--cerebellar cell cocultures were prepared by plating 2 × 10~ 6-day-old embryonic spinal cord or 10-day-o~d embryonic cerebellar cells in the presetlce of I0 '-~ M araC on 80-90~'0 fiJ,ed myotub,.+ cultures. Muscle-spinal cord glial cell coculturcs were prepared ~,~,, !,lating t,,~ic,., pa~k~Ud 6-day-old spinal cord background cells (2 × 106 cells) on m 19tuh.e culture.',. Spinal cord- Iibr 9blast cot:ultures were prepared by platix.g 2 × 1(/' 6-day-old dissociated spi~ml cord cells on confluent twice passaged muscle derivcl fibroblasts. (~yclic nucleotic~e levels were determined in triplicate at ,elected time intcrval.~. Cuhurc. were washed wil.h 4 '< I mi o,~ HEt>ES baft'c~cd baiinced ¢.a'.t ~o:ution, pll 7.2. Trichloroacctk acid fl'CA. 0.5 rnl of 10W~} ~;'a', addcd~ the cell.,, scr ~ped from the ~.i~hcs arid the resultant suspension allowed to sit at xoom tem!~erature for 20 rain. The tissue ard solution were then separated by certrifugation. The TCA solution was extracted with 4 × I ml of H20 saturated ether, l!,ophilized, and the residue dissolved in 50 mM sodium acetate buffer, pH 6.2./\t,.'quots ,,¢ere processed by radioimmunoas:ay. The tissue pellets were dissolved ia ~ N NaOH and prc't:in determined by the method of Lo~ry et al. [101. cAMP and cGMP levels were determined in 6-day-old dissuxciated spinal cord and post.fusion muscle ,:ultures grown separately. Bolh remainec: nearly constant in two separate experimer,ts over 9 days in culture. SoinaI cord cultures contained 0.674 + 0.10J pmol/mg protein cAMP 0nea.n + :LD.) ar~:l 0.025 ± 0.CI l pmol/mg proteip _'GMP while muscle contai'aed 0.316 +_ 0.196 p m ~ / m g pr,Aeitt cAMP and 0.023 +~:'.'..P,! '~ pr,.ao~/mg prc, t~zin cGMP. LJpon ce,ct:,ltr.re of spJn~J cord and muscle cells a 4.9 ± 2.9 (~. = 5)-~oid increase in cCMP ~evels was ot~se.,-ved on day 4 rclatNe to day L, The ravage ,~,'as 2.8--8.6-fold. cA~,,4P ~evels remaiu~.~d nearly cot:stunt over the same ti,ac baterva! (Fig. I~). While 4 days w~~sa conve~::ent ti~.e httcfval or.: r which to detefraic~e the m~gaitude of the

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increase, a d d i f i o n N ~ l o n g e r e×p~fiments indicate a c o n t i n u i n g increase i~ c G M P after t 1 days h~ c u k u r e when th~ c G M P ~ , otmn. Onc~ again, c A M P lew@ were v"lr~ua|ty ,:. . . . .m v a n a m a~ ~ 4.27° :~: 4-7 p m o V m g Notein.. T h e i n c r e a ~ in e G M P a~>ears specific for spinal cord neutrons cocm~mYed with m u s ¢ ~ as the ¢ G M P conten< ;~.f b o t h 10,day .cerebel~ar t o E - m u s c l e cocult~res and 6-day spinM ~')rd gha]~ ceD-~, a~,de cocu~tures did not vary s~g ~l~¢aatiy Dye~ 8 to a.} days in culture, Cocultures o f spi~mt cor~ co}Is and muscle t~suai~y s.ar~ t ° {o twitch after ~a :a and have been observed to eor~ti'~ue ~wi{ching for up to ~1 days. Since the~e aas a md the increase i~- c G M P enaptic tranamissiar} was aic acety~ehdine "eceptor sodiumodependent action aspects o f the increase in jrowt~ i~~ the pro>once of the in~:rease of m d y on el,her cle contacts are k n o w n m f o r m in the i~creaae in c G M P m a y reflect an a~te~ na~e m ~ o f e o m m ~ m ~ t m n relevant to synaptogenesis° ~o~i¢ tissxle

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lumbar sl:.~na~ cord and dor-ial thigh mu~le dissected from 5-, 6-, % and 8-day-old embryos (Fig. Ib). eGMP levels r~-main~ nearly constant in muscle at 0.059 _ 0.C.08 pmol/mg protein while in spinal cord there was a 5. l.-feld increase from 0.085 pmol/mg protein at 5 days to 0.357 pmot/rag protein at ~, "~ . . . . " ~ ' r~,ult clearly demor~.strates a marked nerease in cGMP levels in spinal cord ~ and not mu:cle d~ ring the initial phase of neuromuscular junction formation in vivo. In addition, this experiment ~ggests the lxassil)ility that contact between the axon ~er~ir, al ant~ target muscle fiber, initiate the . ~ M P inerear,e within the terminal and that retrograde axopiasmic tran~pu,,'t accounts for its apparent accumulation in the cell bodies. This is s~apported in part by thr observation that brain :',yna~osornes were found to be eariched in guanylate cyciase [14.]. Alternativdy, a signal other than cGMIa could be produced in the terminal by either muscle contact or association, which via retrograde axoplasmic transport induces gua:~ylate cyclase acti,ei:y within the cell body resulting in increased soma cGMP levels. In a correlate dr;sue cu|~ure experiment, 6-day-old spinal cord ~xplants were plated on mature, muscle cultures. At appropriate times the explants were removed and cGMP levels in the cord explants and cultured mt scle determined separately. As with the in vivo exp,,.-riment cGMP levels remained nearly constant in muscle at 0.075 + (}.()a,. pmol/mg protein over I0 days (F~g. 2a] while level~ in the spinal cord explants increased 2.3-1"otd from 0.257 to 0.598 pmol/mg protein. A~ with the in vivo cxp:riment the increase in cGMP in culture takes place in the sp:nal cord ant~ not in the muscle. At, addilif,~ml question of interest is whether the increa~ it~ neuronal of.iMP i,; mr.OLd,ted by cataract with a tnuscl¢ cell cr by azsoci~lion with a difhtsible mu.~clc i

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sociation between ~t cord cd!s thin is ;ivo. In cukure the independent of a ependent of either ;ecreted by muscle he morea, in cGMP, {t would be premature to Specta:late on thb- in the absence of add~t~onm dat~., as

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2~ 3 Hail, Z.W. x~d Kel!:', R.B.. E~zymatic d,e~.c~m'~.m of endplate a~e~ytd'~.lm.~;s,-.,-r~,efrc,m musc|e, Nature New B;ot.. 2:]2 slr., 4 (19"/g) 374. I(~ R~=hin, i .I.., Schuet/c. ~.M, Wcill. ( 1. and l~tschbach, G.D., Regulation of a,cetyl~ hoiine,'era,,e apF,car~nce al net~ronv.t:cular jtmct~on~, in vitro, Nature (Lond.L 283 fl980) 264--267. 17 Schnaat, R.L. and .",k', .ttncr, A . i : . . Sops.ration of call type,, from embryonic ch~ck a vl rat ~.pinal co..l: ~.l:;,.racteiizatio. o+ ,n.t.t~,c,s.Jn ~:n~ich*:d fiat:hm,,,, J. N~:uror,ci., 1 (1981j 2{.H,--. 17. 18 Wilxon, ILW., P,;ielwL~: P.%. W:d~,+cr, t" R., l.iuk:~,r+, "I.A. and Fry, ,M.r),, ;+ro,t.Cuoior ;:nd rclea;c of acet:,,choline,;tc,a,... ;~v cuhured chick embryo mu~;,:le, Develop. Biol., 33 (19'~g) 285-299.