Molecular Brain Research

8/14/2019 Molecular Brain Research

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E L S E V I E R Molecular Brain Research 24 (1994) 107-117

MOLECULARBRA IN

RESEARCH

R e s e a r c h R e p o r tD e n o v o s y n t h e s i s o f G A P ' 4 3 " i n s i t u h y b r i d i z a t i o n h i s t o c h e m i s t r y

a n d l i g h t a n d e l e c t r o n m i c r o s c o p y i m m u n o c y t o c h e m i c a l s t u d i e si n r e g e n e r a t i n g m o t o r n e u r o n s o f c r a n i a l n e r v e n u c l e i i n t h e r a t b r a i n

G . P a l a c i o s a ,, , G . M e n g o d b , M . S a r a s a c , j . B a u d i e r a , J . M . P a l a c i o s ba Department of Cellular Biology and Physiology, Subunit of Histology, Faculty of Medicine, Universidad Autdnoma de Barcelona, Bellaterra,Barcelona 08193, Spain, b Department of Neurochemistry, CID-CSIC, Barcelona, Spain, c Department of Anatomy, Embryology and Genetics,Veterinary Faculty, University of Zaragoza, Zaragoza, Spain, a Laboratoire de Biologie Moleculaire, Cycle Cellulaire, INSERM U309,38041 Grenoble, France

(Accepted 28 December 1993)

A b s t r a c t

In order to investigate the modulat ion of the synthesis and the subcellular localization of the growth associated proteinGAP-43 in neuronal cell bodies we have taken advantage of the well known regenerative properties of axotomized motorneurons of the facial and hypoglossal nuclei. Alterations in the levels of GAP-43 mRNA containing cells were studied by in situhybridization histochemistry. The protein localization was examined using immunohistochemistry at the light and electronmicroscopic levels. Neurons from the contro l side showed undetectable levels of both GAP-43-1ike immunoreactivity andGAP-43 mRN A levels. Whereas axotomized neurons exhibited a marked increase in GAP-43 mRN A levels and in GAP-43-1ikeimmunoreactivity. Three to 50 days after axotomy, motor neurons ipsilateral to the lesion displayed a dense reticular o rfilamentous perinuclear distribution of the imrnunoreactivity in somata and proximal dendri tic processes, corresponding to thelocation of the Golgi apparatus in these neurons. At the electron microscopic level the immunoreactivity was located in thecisternae o f the Golgi complex and found to be associated with trans-side vesicles of these complexes. The myelinated fibers ofthe transectomized facial nerve also presented an intense GAP-43-1ike immunoreactivity. Twenty-one days after the axotomy adecay in the number of immunostained neurons and in the intensity of immunolabeled somata was observed. Our s tudy reveals arapid induction of GAP-43 mRNA and protein after axotomy. The localization of the newly synthesized GAP-43-1ikeimmunoreactivity to the Golgi apparatus seen in the present work suggests an early association of this protein with newly formedmembranes prior to transport toward the terminals through the axons.Key words: GAP-43; Rat brain; Immunocytochemistry; mRNA; Axotomy; Facial nucleus

1 . I n t r o d u c t i o n

Growth-associated protein GAP-43 (B-50, F1, pp. 46and neuromodulin) is a 24 kDa neuron specific phos-phoprotein that has been implicated in the axogenesisand synaptogenesis during the develop ment and regen-eration of the nervous system [3,4,7,9,14,25,43,45,48,49,51,53]. The levels of GAP-43 are increased duringthe postnatal period in the rat brain [4,14,22,40,41,53],whereas in the adult brain its concentration declinesrapidly following the establishment of final synaptogen-

* Corresponding author.0169-328X/94/$07.00 1994 Elsevier Science B.V. All rights reservedSSDI 0169-328X(93)00006-Z

esis [2,3,14,20,25,26,44,48,50]. However, the protein andits mRNA persist in moderated levels in certain loca-tions of the mature CNS [5,6,12,14,16,36,42,43] and thishas been correlated with a potential role of GAP-43 insynaptic remodeling and functional plasticity of theseareas throughout life [4,14,53]. GAP-43 is a proteinsynthesized in the neuronal somata, rapidly trans-ported throughout axons and localized in growth conesand synapses [15,27,39,52].

Immunoelectron microscopic studies have demon-strated that the localization of GAP-43 is predomi-nantly within axon terminals, small unmyelinated andthin myelinated axons, as well as in dendritic spines[17,59,60]. Following axotom y of peripher al nerves (sci-


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108 G. Palacios et aL / Molecular Brain Research 24 (1994) 107-117

a t i c and fac ia l ne rves ) dramat ic inc reases in GAP-43e x p r e s s i o n h a v e b e e n d e s c r i b e d i n a x o t o m i z e d n e u r o n s[4,8,13,24,29,45,50,53,56,57,63,64]. This increase in boths y n th e s is a n d t r a n s p o r t o f G A P - 4 3 h a s b e e n c o r r e l a t e dwi th the ab i l i ty of axons to regenera te . Immunocyto-chemica l s tudies of the axoto mized neu rons have a l sodemons t ra ted tha t GAP-43 i s a s soc ia ted wi th unmyei i -n a t e d a n d m y e l i n a t e d r e g e n e r a t i n g a x o n s a n d w i t he x t r a a x o n a l e l e m e n t s o f p e r i p h e r a l n e r v e s su c h a sSchwann ce l l s [11 ,56 ,66] . In two recent immunocyto-chemica l s tudies [47,55] accumula t ion of GAP-43 hasb e e n d e s c r i b e d i n n e u r o n a l s o m a t a o f d o r s al r o o t g a n -gl ion a f t e r pe r iphera l ne rve c rush or resec t ion .

In v iew of the ev idence tha t GAP-4 3 may be as soci -a ted wi th regene ra t ive response , e spec ia l ly in ex t r ins icneurons of the ne rvous sys tem, we have examined thedi s t r ibut ion of th i s pro te in by l ight and e lec t ron mi -c roscopy in axotomized fac ia l nuc leus us ing wel l char -ac te r i zed monoc lona l and polyc lona l an t ibodies . Toobta in a more comple te p ic ture we have a l so de te r -m i n e d t h e l o c a t i o n o f c e ll s c o n t ai n i n g m R N A c o d i n gfor i t by in s i tu hybridizat ion his tochemis try us ing radi-o labe led o l igonuc leot ides as probes .

32p]dA T P (3000 C i /m m ol , N E N ) and t e rm ina l deoxynuc leo t idy l -t r ans f e r as e (B oehr inge r M a nhe i m ) to s pec i f i c ac tiv i t i es o f 019 -210104 C i /m m ol . L abe led p robes w ere pu r i f i ed by ch rom atog raphyt h r o u g h a N A C S P R E P A C c o l u m n ( B R L ) .2.2.2. Tissue preparation

T is s ue s ec t ions f rom les ioned an im als , s ac r i f i ced a t s evera l pos t -opera t ion t im es , w ere a l l hyb r id ized a t the s am e t im e . A t l eas t tw os ec t ions pe r l eve l and an im al w ere u s ed . H ow ever , no t in a l l cas esw as i t pos s ib le to ob ta in exac t ly the s am e leve l o f the hypog los s a lnuc leus ( s ee F ig . 2) . F rozen t i s s ue s ec t ions w ere thaw e d to ro omtem pera tu re , a i r d r i ed , and f ixed by im m ers ion fo r 20 m in in 4%p a r a f o r m a l d e h y d e i n P B S ( 2 .6 m M K C I / 1 . 4 m M K H 2 P O 4 / 8 m MN a 2 H P O 4 / 1 3 6 m M N a C I ) , w a s h e d o n c e in 3 P B S a n d t w ic e i n1 P BS , 5 m in each . T h ey w ere in cuba ted in a f res h ly p repareds o lu t ion o f p r e -d iges ted p ronas e a t a f ina l concen t r a t ion o f 24 U /m lin 50 m M T r i s -H CI pH 7 .5 , 5 m M E D T A fo r 10 m in . T he p ro teo ly t i cactivity w as s topped by im m ers ion in a s o lu t ion o f 2 m g / m l g !ycine in

2 . M ate r ia l s and m e thods

2.1. Animals, surgery and tissue processingA d u l t , m a l e S p r a g u e - D a w l e y r a t s w e r e u s e d . T h e a n i m a l s w e r e

deep ly anaes t he t i zed w i th s od ium pen tobarb i t a l ( 60 m g /k g , i .p . ). I none g roup o f an im als the r igh t hypog los s a l ne rve w as t r ans ec ted nearthe hypogh) s s a l canal , c lo s e to the o r ig in o f the des cend in g b ranch tothe hypog los s a l ans a . A n im als w ere k i l l ed by decap i ta t ion 6 , 14 h and1, 2 , 4 , 7 , 15, 30 and 60 days af ter the nerve transection and theb ra ins w ere qu ick ly r em oved , f rozen on d ry i ce and s to r ed a t - 2 0 Cun t i l u s ed fo r in s i tu hyb r id iza t ion h i s toc hem is t ry exper im en ts . Coro -na l s ec t ions (20 / zm th ick ) f rom the b ra in s t em w ere ob ta ine d in am icro tom e c ryos ta t (L e i t z 1720 , W etz la r , F RG ) , thaw -m oun ted on toge la t in - coa ted s l ides , and kep t a t - 20C un t i l u s e . I n ano ther g roupof an im als the r igh t f ac ia l ne rve w as cu t nea r i t s ex i t f rom thes tylom astoid fo rame n. A t 3 , 5 , 7 , 14 and 21 days fol lowing nervet r ans ec t ion t he an im al s w ere e i the r k i l l ed and the i r b r a in s f rozen a t- 2 0 C fo r in si tu hyb r id iza t ion exper im e n ts o r w ere pe r fus ed t r an -s ca rd ial ly under e the r an aes thes i a w ith 50 -100 m l o f 0 .9% s a l inefo l low ed by 4% para fo rm alde hyde , 0 .1% g lu ta r a ldehyde and 15%s a tu ra t ed p ic r i c ac id in 0 .1 M pho s pha te bu f f e r (P B) pH 7 .4 . Bra insw ere r em oved and fu r the r f ixed fo r 4 h at 4 C w i th 4%para fo rm aldehyde in 0 .1M P B, and then w ere s to r ed overn igh t in 5%s ucros e in 0 .1M P B a t 4 C . Corona l s ec t ions (40 ~m ) w ere ob ta inedw i th a v ib ra tom e (L ancer ) a t the nuc leus f ac ia l i s l eve l . T he s ec t ionsw ere then w as hed fo r 12 -20h in 5% s ucros e in 0 .1 M P B a t 4 C .2.2. In situ hybridization procedures2.2.1. Probes

O ligonuc l eo t ide p robe s w ere s yn thes ized on a 380B A pp l iedB i o s y s t e m s D N A s y n t h e s iz e r a n d p u r i f i e d o n a 2 0 % a c r y l a m i d e / 8 Mu r e a p r e p a r a t i v e s e q u e n c i n g g e l. T h e o l i g o m e r s w er e c o m p l e m e n t a r yt o b a s e s 3 0 0 - 3 5 0 ( g a p 4 3 / I ) a n d 5 5 0 - 6 0 0 ( g a p 4 3 / I I ) o f t h e r a tG A P -43 cD N A [28 ]. T hey w ere l abe led a t the i r 3 ' end w i th [ a -

B dF ig. 1 . E xpres s ion o f G A P -43 m R N A af te r 15 days o f f ac ia l ne rvet r ans ec t ion . A : T o lu id ine - s ta ined s ec t ion . B : pho tom icrog raph o f anau to rad iog ram o f a r a t s ec t ion hybr id ized w i th a 32P -labe led o l igonu-c l e o ti d e c o m p l e m e n t a r y t o t h e r a t G A P - 4 3 m R N A . D a r k a r e a sco r r es pond to r eg ions r i ch in hyb r id iza t ion s igna l. T he g ra nu le ce l l so f the ce r ebe l lum s how an in tens e hybr id iza t ion s igna l . T he a r row spo in t the l e s ioned n uc leus f ac ia l i s. T he a r row heads po in t to theips i l a te r a l s ide . O bs erve the d ram at ic inc reas e in the hybr id iza t ions igna l o f the m o to r neu rons o f the nuc leus f ac ia l i s in the l e s ionedneurons . Bar - - 2 m m .


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G. Palacios et aL / Molecular Brain Research 24 (1994) 107-117 10 9P BS du r ing 30 s . T he s l ides w ere th en r in s ed tw ice in P BS fo r 30 s ,and dehydr a ted in a g r aded s e r i es o f e thano l (60 -100%) .2.2.3. Hybridization

L abe led p ro bes w ere d i lu ted to a f ina l concen t r a t ion o f 0 .4 -0 .8p m o l / m l i n 5 0% f o r m a m i d e , 6 0 0 m M N a C l , 1 0 m M T r i s - H C l p H7 .5 , 1 m M E D T A , 1 D en hard t ' s s o lu t ion (0 .02% F ico l l, 0 . 02%polyv iny lpy r ro l idone , 0 .02% bov ine s e rum a lbum in ) and 500 / zg / ,~ l

yeas t tRN A . T i s s ues w ere cove red w i th 60 p . l o f hyb r id iza t ions o lu t ion , over la id w i th nes co f i lm cover s l ip s , and hybr id ized fo r 17 hin a hum id cham ber a t 42C . T he s ec t ions w ere t hen w as hed a t 50Cin 600 m M N aCI , 10 m M T r i s -H Cl pH 7 ,5, 1 m M E D T A fo r 4 h w i th4 changes o f bu f f e r . T i s s ues w ere de hydra t ed and e i the r app os ed to/ 3 -m a x f i lm ( A m e r s h a m ) o r d i p p e d i n t o A m e r s h a m L M - 1 n u c l e a rt r ack em uls ion . F i lm s w ere deve loped a f t e r 4 days and em uls ionaf te r 7 days in K odak D -19 . T i s s u es w ere s t a ine d w i th G iem s a ,

F ig . 2 . E f f ec t o f the t r ans ec t ion o f the hypog los s a l ne rve on the l eve l s o f G A P -43 m RN A a t d i f f e r en t t im e s a f t e r l e s ion . A r row s po in t ou t thenuc leus o f the l e s ioned nerve . P ic tu res a r e pho tom icro g raph s f rom f i lm au to ra d iog ra m s genera ted f rom t i s s ue s ec t ions hybr id ized in the s am eexper im en t . T h e s ec t ions do no t co r r e s pond to exac t ly the s am e leve l o f the hypog los s a l nuc le i . N o te th e f as t inc r eas e in the l eve l s o f G A P -4 3m R N A (hybr id iza t ion s igna l can be obs e rv ed a l r eady a t 14 h ) and the r e tu rn to bas a l l eve l s a f t e r s u rv iva l t im es o f 60 days. Bar = 2 m m .


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110 G. Palacios et aL /Mo lecu lar Brain Research 24 (1994) 10 7-117dehydrated and mounted with Eukitt. Sections were examined usinglight- and dark-field microscopy (Leitz, Orthoplan).

The specificity of the oligonucleotide probes was assessed bydifferent criteria (Sarasa et al., in preparation). By Northern blotanalysis on total RNA extracted from several regions of the rat brain,a single mRNA species of approximately 1400 nucleotides was de-tected with a regional distribution similar to the one observed in thein situ hybridization experiments. Two oligonucleotides complemen-tary to different regions of the GAP-43 mRN A were used independ-ently as hybridization probes in consecutive sections from the sameanimal and showed an identical pattern of hybridization. Cohy-bridization of a labeled oligonucleotide with a 20-fold excess of thesame unlabeled probe resulted in the complete abolishment of thehybridization signal. The signals were n ot af fected when the oligonu-cleotide added in excess was complementary to a different region ofthe same mRNA. The thermal stability of the hybrids was deter-mined by washing at increasing temperatures: a sharp decrease inthe hybridization signal was observed at a temperature consistentwith the theoretical value of melting temperature (Tm) [38] of thehybrids formed.

2.3.2. lmmuno-light and -electron microscopyTissue sections were first incubated with either GAP-43 mono-

clonal antibody (diluted 1:1000) or GAP-43 polyclonal antibody(diluted 1:400) for 17 h, rinsed and in cubated with the reage nts ofthe avid in-biotin-pero xidase kits (mouse Vectastain ABC and rabbitVectastain ABC kits respectively, Vector Laboratories) using recom-mende d dilutions. Peroxidase activity was finally visualized by incu-bating t he sectio ns with 0.05% 3,3'-diamino benzidine and 0.01%H2 0 2 in PB for 5-10 min. Some sections were processed with1-naphtol solution for 2 min [35]. For electron microscopic observa-tions, blocks of the facial nucleus obtained with punches of themonoclonal antibody tre ated material we re postfixed in 1% OsO 4 for1 h, block-stained in 1% uranyl acetate veronal buffer, dehydratedand fla t-embe dded in Durcup an (Fluka). Semithin sections (1 ~m)were cut on an LKB Ultratome III and stained with 1% ToluidineBlue for light-microscope photography. Ultrathin sections were alsoobtained and stained with alcoholic uranyl acetate for I0 min or withuranyl acetate and lead citrate for 5 min. They were later examinedand pho togra phed with a Hitachi H-7000 electron microscope.

2.3. Immunocytochemical procedures2.3.1. Antibodies

GAP-43 monoclonal antibody was obtained from BoehringerManheim (Germany) and the GAP-43 polyclonal antibody was ob-tained by immunizing rabbits with GAP-43 purified from bovinebrain. The antibody production, purification and characterization ofthe l atter antibody have been describe d previously [1,34]. Theavidin-biotin-peroxidase pre-embedding technique was used for lightand electron microscopic localization of both GAP-43 antibodies.

3. Results

3 . 1. I n s it u h y b r i d i z a t i o n h i s t o c h e m i s t r yT h e o l i g o n u c l e o t i d e p r o b e s u s e d i n t h e s e e x p e r i -

m e n t s , r e v e a l e d a d i s t r i b u t i o n o f G A P - 4 3 m R N At h r o u g h o u t t h e r a t b r a i n w h i c h i s i n c o m p l e t e a g r e e -m ent wi th p rev iou s pub l i ca t io ns [9, 31]. High l eve l s o f

Fig. 3. Cellular localization of GAP-43 mRNA in the hylgoglossal nucleus 2 days after the nerve transection. The autoradiographic image ispresented in A as a dark-field photomicrograph from emulsion-dipped tissue section in which autoradiographic grains are seen as bright points.Only lesioned neurons express GAP-43 mRNA. The arrows point out the same neuron in both fields. B is a bright-field image of the sectionshown in A. Bar = 200/zm.


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G. Palacios et al. / Molecular Brain Research 24 (1994) 107 -117 111

h y b r i d i z a t i o n s i g n a l w e r e s e e n i n c o r te x , h i p p o c a m p u s ,s o m e t h a l a m i c a n d h y p o t h a l a m i c n u c l e i , l o c u sc o e r u l e u s , d o r s a l a n d m e d i a l r a p h 6 , g r a n u l e c e l l s o f t h e

c e r e b e l l u m , a n d n u c l e i o f m e d u l l a o b l o n g a t a ( S a r a s a e ta l, m a n u s c r i p t i n p r e p a r a t i o n ) . F u r t h e r m o r e , c o n t r o le x p e r i m e n t s ( s e e M a t e r i a l s a n d m e t h o d s s e c t i o n )

Fig. 4. Light microscopic immunoreactivity in facial nuclei using a polyclonal antibody to GAP-43. A,B: 3 days following axotomy. C,D: 2l daysfollowing axotomy. A: vibratome section of the ipsilateral facial nucleus showing a intense im munor eactivity in the som ata an d pro ximaldendri tes of motor neurons (arrows). B: the motor neurons o f the contralateral facial nucleus are devoid of immunolabel ing (arrows). C:axotomized motor neurons at higher magnificat ion showing a perinuclear dense and ret icular dist ribut ion of the immunoreact ivi ty (arrows).Other neurons exhibi ted a reduced intensi ty of the immunolabel ing (arrow heads). Immunostained axons and fiber bundles can be seen in theneuropi l . Note the ausence of terminals on the plasmalemma of axotomized neurons. D: contralateral facial motor neurons are devoid ofimmunoreactivi ty. Note the immunostained axons and fiber bundles in the neuropi l . Imm unolabel ing boton terminals can be observed in contactwith the plasma lemma o f motor neuro ns (small arrows). B ar = 100 ~zm in A, B; 20 /xm in C, D.


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112 G. Palacios et al. /Molecular Brain Research 24 (1994) 107-117

A


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G. Palacios et aL /Molecular Brain Research 24 (1994) 107-117 113proved the specificity of the hybridization signal ob-tained with the labeled oligonucleotide probe (data notshown). In unoperated, sham operated and in the sidecontralateral to the lesioned side, GAP-43 mRNA wasundetectable in both facial and hypoglossal nerve nu-clei. Fifteen days after unilateral transection of thefacial nucleus, the concentration of GAP-43 mRNA inthe lesioned motor neurons increased in a dramaticmanner (Fig. 1B) when compared to the basal levelsseen in the contralateral unlesioned side. As early as14 h after transection, GAP-43 mRNA can be detectedby in situ hybridization (not shown).

Unilateral axotomy of the hypoglossal nerve re sultedalso in a prominent increase in the levels of GAP-43mRNA when compared to the contralateral unlesionedside (Fig. 2). This rise was already evident 14 h afterthe lesion (Fig. 2) The highest levels were observedfrom 48 h onwards, beginning to decline by 30 daysonwards, presumably when reinnervation is takingplace, to reach control levels 60 days after the lesion.3.2. Cellular localization of G AP-43 m RN A

Nuclear emulsion autoradiography shows that thelabeled hybrids are located exclusively in the lesionedmotor neurons of the facial (not shown) and hypoglos-sal (Fig. 3) nuclei 2 days after the nerve transection.3.3. Immuno cytochemistry: light microscopy

of immunolabeling in some of them was, however,reduced 21 days after nerve transection (Fig. 4C).At higher magnification the staining pattern in thecell bodies of the axotomized neurons differed slightlywith the two antibodies used. With the polyclonalantibody, the cytoplasm exhibited a dense homoge-neous or reticular distribution of the immunoreactiveproduct surrounding the unstained nucleus (Fig. 4C).The monoclonal antibody revealed thin filamentouspatches of the immunoreactive product distributed in aperinuclear manner, in agreement with the well-knowndistribution of the Golgi apparatus in these motorneurons (Fig. 5C). The neuropil in the contralateralfacial nucleus showed immunostained nerve processesand nerve terminals. Most of these synaptic terminalswere observed covering the perikarya and main den-drites of motor neurons (Figs. 4D and 5D). On thetransected side, the motor neurons in the facial nu-cleus appeared free of these synaptic contacts probablydue to the fact that the surface area of motor neuronswas occupied by microglial activated cells which dis-placed these synaptic bouto ns (synaptic stripping) (Figs.4C and 5C). The facial nerve in the ipsilateral sideshowed an intense immunoreactivity in regeneratin gmyelinated fibers (Fig. 5A). In contrast, the contralat-eral facial nerve appeared unstained. (Fig. 5B).

3.4. Immuno cytochemistry: electron microscopyThe two antibodies used in the present study dis-

played a high degree of immunoreactive sensitivity.This immunoreactivity was predominantly restricted tothe neuropil and axon bundles of the brainstem zoneand facial nuclei while the neuronal somata were un-stained (Figs. 4B,D and 5D). The immunosta ining sig-nal in the neuropil was stronger when the monoclonalantibody was used (Fig. 5C,D).

Three days after unilateral transection of the rightfacial nerve, the motor neurons and their proximaldendritic processes of the contralateral facial nucleusappeared unstained and surrounded by a homogeneousdense labeling of t he neuropil (Fig. 4B). In contrast, anincrease in GAP-43-1ike immunoreactivity in moto rneurons was seen in the ipsilateral facial nucleus (Fig.4A). In the following days examined (5-15 days) thevast majority of the motor neurons in the ipsilateralside showed GAP-43-1ike immunoreactivi ty in theirsomata. The number o f labe led cells and the intensity

The results of electron microscopic immunocyto-chemistry were in accordance with previous light mi-croscopic studies of semithin sections (Fig. 6A,B).These sections revealed a perinuclear pattern of densepatches restricted to motor neurons in the ipsilateralside (Fig. 6A). The electron microscopic study showedthat the immunoreactivity was associated with the dis-persed Golgi complexes (rete dispersion) only in thecytoplasm of axotomized neurons (Fig. 6C). These in-jured motor neurons were covered by activated mi-croglial cells (Fig. 6C). At higher magnification, theimmunoreaction product was scattered between thecisternae stacks throughout the Golgi complex (Fig.6D,E). Some lightly immunoreactive vesicles were alsoseen in the trans-side of the Golgi complex (Fig. 6E).Labeling of other cellular organelles in these injuredneurons was not observed. Attempts to furthe r localizeGAP-43 using immunogold techniques, which has beenmainly used in cultured neurons, failed in our tissues.

Fig. 5. Light micro scopic immunoreactivity in facial nuclei using a monocl onal antibody to GAP-43, 7 days following axotomy. A: vi brat omesection of the ipsilateral facial nerve exhibiting a intense immunoreactivity in myelinated fibers comparing with the absence of immunoreactivityin contralateral facial nerve shown in B. C: axotomized motor neurons shows perinuclear filamentous immunoreactive patches in the citoplasmcorresponding o the Golgi apparatus localization (small arrows). D: contralateral facial motor neurons are devoid of immunoreactivity (arrows).Note the dense immunoreaction product in the neuropil and terminals surrounding these neurons. Bar = 40/ zm in A, B; 25/ zm in C, D.


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G. Palacios et al./M olec ular Brain Research 24 (1994) 107- 117

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G . Pa lac io s e t a l . /M o lec u la r B r a in Res ear ch 24 ( 1994 ) 107 - 117 11 54 . D i s c u s s i o n

The present study shows that a rapid increase inGAP-43 mRNA levels and accumulation of GAP-43 inmotor neurons cell bodies and their proximal axonsegments occur after nerve axotomy. This initial accu-mulation remained at a high level until 3 to 4 weeksafter lesion, when it began to decline. By immunoelec-tron microscopy we have been able to show that motorneurons in the lesioned side exhibited considerableGAP-43-1ike immunoreactivity in the cell bodies andthat the newly synthesized protein was localized in theGolgi apparatus. While increases in GAP-43 mRNA inspinal motor neurons [9,32] and in the nucleus facialis[46] have been already shown, this is the first study inhypoglossal neurons. Furthermore, we show a quiteearly increase, demonstrable as short as at 14 h afterlesion.

The fact that both GAP-43 mRNA and protein arebelow detection levels in the resting 'normal' neuronsand increase dramatically after axotomy is consistentwith an induction of the de novo synthesis and trans-port of the protein throug hout injured axons, eventsthat can be modulated by the well-known regenerativepotentia l of the facial and hypoglossal axotomized neu-rons [30]. Facial neurons and other PNS neurons havethe capacity to regenerate while the CNS neurons donot. This has been correlated with the incapacity ofthese CNS neurons to express regeneration-associatedproteins like GAP-43 [53,57]. However, in some centralneurons such as rubrospinal and in retinal ganglionneurons, GAP-43 mRNA can be expressed after axo-tomy [18,20,33,57].

GAP-43 has been proposed as a protein normallydisposed on the cytoplasmic surface of axonal mem-branes [3,21,37,58]. The binding of this protein to thevesicular elements in the Golgi apparatus can facilitatethe transport to the distal axon segments and theirattachment to the cytoplasmic side of membranes. Inthis sense, other studies have demonstrated a subcellu-lar localization of GAP-43 in vesicle and plasma mem-branes [17].GAP-43 has been ultrastructurally localized in un-myelinated and thinly myelinated axons of the adultpyramidal tract [21]. In the rat neostriatum, GAP-43was distributed in discrete patches throughout fine

caliber unmyelinated axons and in axospinous asym-metric synapses [17]. GAP-43 immunoe lectron reactiv-ity has been also found in the neostriatal neuropildistributed in cytoplasmic patches in dendrites, den-dritic protrusions and thin spines [17]. Rece nt lightmicroscopic immunocytochemical studies showed accu-mulation of GAP-43 in the cell bodies of dorsal rootganglion neurons (DRG) after sciatic nerve crush orresection [47,55]. 100% of L 4 and L 5 DRG neuronsexpressed and accumulated detectable amounts ofGAP-43 in their cell bodies 1 week after sciatic nerveinjury [47].

Previous studies [23], have suggested that GAP-43 isconcentrated in the Golgi apparatus during the devel-opmental stages of hippocampal neurons, by compar-ing the immunocytochemical distribution of this pro-tein with that of wheat germ agglutinin, a lectin thatselectively labels this organelle. Meiri and coworkers[40] have also noted a perinuclear concentration ofGAP-43 in cultured sympathetic neurons compatiblewith its localization to the Golgi apparatus.The majority of electron microscopy studies withGAP-43 have focused on the location of this protein ingrowth cones, axons and terminals in the adult animal[17,21,58,65]. These studies have not provided evidenceas to where in the cell body this protein could besynthesized. Biochemical studies have proposed thatGAP-43 is actively synthesized as a soluble proteinwhich rapidly associates with membranes [10,54]. AsGAP-43 is not a glycoprotein, fatty acylation has beenproposed as the mechanism for its membrane attach-ment [10,54]. More recently, GAP-43 immunoreactivi tyhas been located using immunogold probes, on thecytosolic face of electron-lucent putative transpor t vesi-cles in the trans region of the Golgi apparatus, inneurites and in growth cones of cultured hippocampalneurons [61]. Quantification of the density of GAP-43on the plasma membrane of hippocampal pyramidalneurons demonstrated no differences between growthcones and neuritic processes, which may indicate thatin in vitro conditions, GAP-43 does not play a selectiverole in growth cones [62].The results in this work show that in vivo and inneurons of the rat cranial nerve, with a well knownregenerative potential, GAP-43 is concentrated in cellbodies, indicating that newly synthesized GAP-43 could

Fig . 6 . A , B : l i g h t m ic r o sco p ic im m u n o r ea c t iv i t y i n sem i th in sec t io n s o f f ac i a l n u c l e i u s in g a m o n o c lo n a l an t ib o d y to GAP - 4 3 , 3 d ay s a f t e ra x o t o m y . A : i n j u r e d m o t o r n e u r o n s d i s p l a y i m m u n o r e a c t i v e p r o d u c t d i s p e r s e d i n d i s c r e t e p a t c h e s i n a p e r i n u c l e a r p o s i t i o n ( s m a l l a r r o w s ) B :c o n t r a l a t e r a l m o t o r n e u r o n s a p p e a r e d w i t h u n l a b e l e d c y t o p l a sm . I n b o t h s i d e s t h e f i b e r s i n t h e n e u r o p i l e x h i b i t e d i n t e n s e i n m u n o r e a c t i v i t y . C , D :e l e c t r o n m i c r o s c o pi c i m m u n o r e a c t i v i ty i n t h i n s e c t i o n s o f t h e s a m e i n j u r e d n e u r o n s s t a i n e d w i t h a l c o h o l ic u r a n y l a c e t a t e . C : a x o t o m i z e d m o t o rn e u r o n s h o w i n g d i s p e rs e d i m m u n o r e a c t i v e G o l g i c o m p l e x i n t h e c y t o p l a s m ( a r ro w s ) . N o t e t h e u n l a b e l e d n u c l e u s ( N ) , n u c l e o l u s (N o ) a n dm i c r o g l i a l a c t i v a t e d c e l l ( M ) a t t a c h e d t o n e u r o n p l a s m a l e m m a . D : i m m u n o r e a c t i v e G o l g i c o m p l e x i n i n j u r e d m o t o r n e u r o n s ( a r r o w s ) . T h er e a c t i o n p r o d u c t a p p e a r s b e t w e e n s t a c k c is t e r n a e ( a rr o w s ) . N o t e a n a c t i v a t e d m i c r o g l ia l c e ll (M ) . E : a x o t o m i z e d m o t o r n e u r o n s t a i n e d w i t hu r a n y l a c e t a t e a n d l e a d c i t r a t e s h o w i n g i m m u n o s t a i n e d r o u n d v e s i c le s lo c a t e d i n t h e t r a n s s i d e o f t h e G o l g i c o m p l e x (a r r ow s ) . B a r s = 2 0 / x m i nA , B ; 1 / z m i n C ; 0 . 5 / x m i n D , E .


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11 6 G. Pa acios et al. / M olecular Brain Research 24 (1994) 10 7-1 17be associated with Golgi sacules and vesicles prior totransport throughout the axons to synaptic terminals.The localization of GAP-43 to the Golgi apparatus, theorganelle involved in the formation of a variety oftransport vesicles and in the traffic of those vesiclesfrom the Golgi to the plasma membrane [19] suggestsan important role of the protein in axonal regenera-tion.

References

[1] Baudier, J . , Bronner, C. , Kl igman, D. and Cole , R.D., Pro te inkinase C subst ra tes from bovine brain . Puri f icat ion and charac-t e r i za t io n o f n eu ro m o d u l i n , a n eu ro n -sp ec i f i c ca l mo d u l i n -b i n d -ing pro te in , J . Bi o L Ch em . , 264 (1989) 1824-1828.

[2] Benow itz , L.I . , Shashona, V .E. and Y oon, M., Speci fic changesin rap id ly t ransported pro te ins during regenerat ion of the gold-fi sh opt ic nerve, J . Neurosci., 1 (1981) 30 0-307.[3] B enowitz , L.I . and Lewis, E.R. , In crease d t ranspor t of 44 ,000-to

49,000- dal ton acid ic pro te ins during regenerat ion of the gold-fi sh opt ic nerve: A two-dimensional gel analysis , J . Neurosci., 3(1983) 2153-216 3.[4 ] Ben o w i t z , L . I . an d Ro u t t en b e rg , A . , A memb ran e p h o sp h o p ro -re in associa ted wi th neural development , axonal regenerat ion ,phosphol ip id metabol i sm and synapt ic p last ic i ty . Trends Neu-rosci., 10 (1987) 527-531.

[5] Benowitz , L.I . , Aposto l ides, P.J . , Perrone-Bizzozero , N.I . , Fin-k leste in , S.P. and Zwiers , H. , Anatomical d is t r ibut ion of theg ro w t h -a sso c i a t ed p ro t e i n G A P-4 3 / B-5 0 i n t h e ad u l t ra t b ra i n ,J. Neurosci., 8 (1988) 339 -352.

[6] Benowitz , L.I . , Perrone-Bizzozero , N.I . , Finkleste in , S.P. andBi rd , E .D . , Lo ca l i za t i o n o f t h e g ro w t h -a sso c i a t ed p h o sp h o p ro -t e i n G A P-4 3 i n t h e h u m an ce reb ra l co r t ex, J . Neurosci., 9 (1989)990-995.[7] Benowitz , L.I . , Rodriguez, W.R. and Neve, R.L. , The pat tern ofG A P-4 3 i mmu n o s t a i n i n g ch an g es i n th e ra t h i p p o camp a l fo rma-t ion during react ive synaptogenesis , Mol. Brain Res., 8 (1990)17-23.[8] Bisby , M.A., Dependence of GAP-43 (B-50, F1) t ransport onaxonal regenerat ion in ra t dorsal root gangl ion neurons, BrainRes., 458 (1988) 157-161.

[9] Chong, M.S. , Fi tzgerald , M., Winter, J . , Hu-Tsai , M., Emson,P .C . , W i ese , U . an d W o o l f , C . J . , G A P-4 3 mRN A i n ra t sp i n a lco rd an d d o rsa l ro o t g an g l i a n eu ro n s : d ev e l o p men t a l ch an g esand re -expression fo l lowing periphera l nerv e injury , Eur. J.Neurosci., 4 (1992) 883-895 .[10] Coggins, P.J . and Zwiers , H. , B-50 (GAP-43): b iochemist ry andfu n c t i o n al n eu ro ch emi s t ry o f a n eu ro n -sp ec i f ic p h o sp h o p ro t e i n .J. Neurochem., 56 (1991) 1095-1106.[11] Curtis, R., Stewart, H.J.S., Hall , S.M., Wilkin, G.P., Mirsky, R.an d Je ssen , K .R . , G A P-4 3 i s ex p re ssed b y n o n my e l i n - fo rmi n gSchwa nn cel l s of the periphe ral ner vous system, J . Cell. BioL,116 (1992) 1455-1464.[12] Curt i s , R , Av eri l l , S. , Priest ley , J .V. and Wilk in , G.P. , T hedist r ibut ion of GAP-43 in normal ra t sp inal cord , J . NeurocytoL,22 (1993) 39-5 0.

[13] Curt i s , R. , Green , D. , Linssay , R.M. and Wilk in , G.P. , Up-regu-l a t i o n o f G A P-4 3 an d g ro w t h o f ax o n s i n ra t sp i n a l co rd a f t e rcompression in jury , J . Neurocytol., 22 (1993) 51-64.[14] Dani , J .W., Armstrong, D.M. and Benowitz , L.I . , Mapping thed ev e l o p men t o f t h e ra t b ra i n b y G A P-4 3 i mmu n o cy t o ch emi s t ry .Neuroscience, 40 (1991) 277-287.

[15] De Graan , P.N.E. , Van Hooff, C.O.M., Ti l ly , B.C. , Oest re icher,A.B., Schotman, P. and Gispen, W.H., Phosphoprote in B-50 inn e rv e g ro w t h co n es f ro m fe t a l ra t b ra i n , Neurosci. Lett ., 61(1985) 235-241.

[16] De la Monte , S.M., Federoff , H.J . , Shi -Chung, Ng. , Grabczyk,E .D . an d F i sh man , M .C ., G A P-4 3 g en e ex p re ss io n d u r i n g d ev e l -opm ent : persis tenc e in a d is tinct ive se t of neurons in the ma turecent ra l nervous system, Dev. Brain Res., 46 (1989) 161-168.[17] Di Fig l ia , M., Roberts , R.C. and Benowitz , L.I . , Immunoreac-t ive GAP-43 in the neuropi l of adul t ra t neost ria tum: local iza-t ion in unmyel inated fibers , axon terminals , and dendri t ic sp ines,J. Comp. Neurol., 302 (1990) 992-1001.[18] Doster, S.K., Lozano, A.M., Aguayo, A3. and Wil lard , M.B.,Expression of the growth-associa ted pro te in GAP-43 in adul t ra tretinal ganglion cells following axon injury, Neuron, 6 (1991)635 -647.

[1 9] Fa rq u h a r , M .G . an d Pa l ad e , G .E . , Th e G o l g i ap pa ra t u s (co m-plex)-(1954-1981)-from artifac t to cen ter stage, Z Cell Biol., 91(1981) 77 s-103s.[20] Freeman, J .A. , Bock, S. , Deaton , M., McGuire , B. , Norden, J .J .and Sn ipes, G.J . , Axonal and g l ia l pro te ins associa ted wi thdevelopment and response to in jury in the ra t and goldfish opt icnerve, Exp. Brain Res. (SuppL), 13 (1986) 34-47.[2 1 ] G o rg e l s , T .G .M.F . , V an Lo o k e ren Camp ag n e , M. , O es t re i ch e r ,A .B . , G r i b n au , A .A .M. an d G i sp en , W .H . , B -5 0 / G A P-4 3 i slocal ized a t the cy toplasmic s ide of the p lasma membrane indeveloping and adul t ra t pyramidal t ract , J . Neurosci., 9 (1989)3861-3869.[22] Gosl in , K. , Schreyer, D.J . , Skene, J .H.P. and Banker, G. , Devel -opment of neuronal polari ty : GAP-43 d is t inguishes axonal fromdendri t ic growth cones, Nature, 336 (1988) 672-674.

[23] Gosl in , K. , Schreyer, D.J . , Skene, J .H.P. and Banker, G. ,Ch an g es i n t h e d i s t r i b u t i o n o f G A P-4 3 d u r i n g t h e d ev e l o p men tof neuronal polari ty , J . Neurosci., 10 (1990) 588-602.

[24] Hoffm an, P.N., Expression of GAP-43, a rap id ly t ransportedgrowth-associa ted pro te in , and c lass II beta tubul in , a s lowlyt ransported cytoskele ta l pro te in , are coordinated in regenerat -ing neurons, J . Neurosci., 9 (1989) 893-897.

[25] Jacobson, R.D., Virag , I . and Skene, J .H.P. , A pro te in associ -a ted wi th axon growth , G AP-4 3, i s widely d is t r ibuted and devel -o p men t a l l y reg u l a t ed i n ra t CN S, Z Neurosci., 6 (1986) 1843-1845.[26] Kalil , K. and Skene, J.H.P., Elevated synthesis of an axonallyt ransported pro te in corre la tes wi th axon outgrowth in normaland in jured pyramidal t racts , J . Neurosci., 6 (1986) 2563-2570.[27] Katz , F. , El li s , L. and P fenninger, K.H., Nerv e growth conesi so l a t ed f ro m fe t a l ra t b ra i n . I I I . Ca l c i u m-d ep en d en t p ro t e i nphosphoryla t ion , J . Neurosci., 5 (1985) 1402-1411.

[28] Karns, L.R . , Ng, S.-C. , Freem an, J .A. and Fishman, M.C.,C l o n i n g o f co mp l emen t a ry D N A fo r G A P-4 3 , a n eu ro n a lg ro w t h - re l a t ed p ro t e i n , Science, 236 (1987) 597-600 .[29] Knyih~ir-Csill ik, E., Csill ik, B. and Oestreicher, A.B., Light andelect ron microscopic local izat ion of B-50 (GAP43) in the ra tsp inal cord during t ransgangl ionic degenerat ive a t rophy andreg en e ra t i o n , J . Neurosci. Res., 32 (1992) 93-109.

[30] Kreutzberg , G.W., Graeber, M.B. and St re i t , W.J. , Neuron-gl ia lre l a t i o n sh i p d u r i n g reg en e ra t i o n o f mo t o r n eu ro n s . MetabolicBrain Dis'., 4 (1989) 81-85.[31] Kruger, L. , Bendot t i , C. , Rivol ta , R. and Samanin , R. Dist r ibu-t i o n o f G A P-4 3 m RN A i n t h e ad u l t ra t b ra in , J . Cornp. Neurol.,333 (1993) 417-434.

[32] Lind , , H. , Piehl , F. , Dagerl ind , ,~ , Verge, V,M.K., Arvidsson,U., Cul lheim, S. , Risl ing , M., Ulfhake, B. and H6kfel t , T. ,Ex p re ss i o n o f G A P-4 3 mRN A i n t h e ad u l t mammal i an sp i n a lco rd u n d e r n o rma l co n d i t i o n s an d a f t e r d i f fe ren t t y p es o f l e -s ions, wi th specia l reference to motoneurons, Exp. Brain Res.,91 (1992) 284-295.


11/11

G. Palacios et al. /M ole cu lar Brain Research 24 (1994) 10 7-1 17 11 7[33] Lozan o, A.M., D oster, S.K., Aguayo, A.J . and Wil lard , M.B.,

Imm u n o reac t i v i ty to G A P-4 3 i n ax o t o mi zed an d reg en e ra t i n gret inal gangl ion cel l s of adul t ra t s , Soc. Neurosci. Abstr., 13(1987) 1389.[34] Masl iah , E. , Mal lory , M., Hansen , L. , Alford , M., Albright , T. ,D e Te re sa , R . , Te r ry , R , , Bau d i e r , J . an d Sa i t o h , T . , Pa t t e rn s o f

ab e r ran t sp ro u t i n g i n A l zh e i mer ' s d i sea se , Neu ro n , 6 (1991)729-739.[35] Ma uro , A. , Germ ano, 1 . , Giaccon e, G. , Giordana, M.T. and

Sch i f fe r , D . , 1 -N ap h t o l b a s i c d y e (1 -N BD ) . A n a l t e rn a t i v e t od i ami n o b en z i d i n e (D A B) i n i mmu n o p e ro x i d ase t e ch n i q u es , His-tochemistry, 83 (1985) 97-102.[36] Mc Gu ire , C.B. , Snipes, G.J . and Norde n, J .J . , Light -m icro-

sco p i c i mmu n o l o ca l i za t i o n o f t h e g ro w t h -a sso c i a t ed p ro t e i nG A P-4 3 i n t h e d ev e l o p i n g b ra in , Dec. Brain Res., 41 (1988)277-291.[37] Mc In tosh , H. , Park inson, D. , Meiri , K. , Daw, N. and Wil lard ,M., A GAP-43-1ike pro te in in cat v isual cortex , l/'ts. Neurosc i., 2(1989) 583-591.

[38] Mein koth ; J . and Wahl , G. , Hyb rid izat ion of nucle ic acidsimmobi l ized on so l id supports , An a l B i o ch em . , 138 (1984) 267-284.[39] Meiri , K.F. , Pfenninger, K.H. and Wil lard , M.B., Growth-associ -a t ed p ro t e i n , G A P-4 3 , a p o l y p ep t i d e t h a t i s i n d u ced w h en

neurons extend axons, i s a component of growth cones andco r re sp o n d s t o p p . 4 6 , a ma j o r p o l y p ep t i d e o f a su b ce l l u l a rfract ion enriched in growth cones, Proc. Nat l. Acad. Sci . USA,83 (1986) 3537-3541.[40] Meiri , K.F. , Wil lard , M. and John son, M .I. , Dist r ibu t ion a ndp h o sp h o ry l a t i o n o f t h e g ro w t h -a sso c i a t ed p ro t e i n G A P-4 3 i nreg en e ra t i n g sy mp a t h e t i c n eu ro n s i n cu l t u re , J . Neurosci., 8(1988) 2571-2581.

[41] Moya, K,L. , Jhaveri , S. , Schneider, G .E. an d B enowitz , L.I . ,Imm u n o h i s t o ch emi ca l l o ca l iza t i o n o f G A P-4 3 i n t h e d ev e l o p i n ghamster re t inofugal pathway, J . Comp. Neurol., 288 (1989) 51-5 8.[42] Neve , R.L. , Finch , E.A., Bi rd , E.D. a nd B enowitz , L.I . , The

growth-associa ted pro te in GAP-43 (B-50, F1) i s expressed se lec-t ively in associa t ive reg ions of the adul t human brain , Proc.Natl. Acad. Sci. USA, 85 (1988) 3638-3642.[4 3] O es t re i ch e r , A .B . an d G i sp en , W .H . , Co m p ar i so n o f t h e i m-mu n o cy t o ch emi ca l d i s t r ib u t i o n o f t h e p h o sp h o p ro t e i n B-50 i nt h e ce reb e l l u m an d h i p p o camp u s o f i mmat u re an d ad u l t ra tbra in , Brain Res., 375 (1986) 267-279.[44] Perry , G.W., Burmeister , D.W. and Grafste in , B. , Fast axonal lyt ran sp o r t ed p ro t e i n s i n reg en e ra t i n g g o l d f i sh o p t i c n e rv e , J .Neurosci., 7 (1987) 792-806.

[45] Redshaw, J .D. and Bisby , M.A., Pro te ins of fast axonal t rans-p o r t i n t h e reg en e ra t i n g h y p o g l o ssa l n e rv e o f t h e ra t , Can. J.Physiol. PharmacoL, 62 (1984) 1387-1393.[46] Saika , T. , Kiyama, H. , Tohyama, M. and Matsunaga, T. , GAP-43m RN A ex p re ss i o n in fac ia l mo t o n eu ro n s d u r i n g reg en e ra t i o n : i nsi tu hybrid izat ion h is tochemist ry using an a lkal ine phosphatase-l ab e l l ed p ro b e , Acta Otolaryngol. Suppl., 501 (1993) 80-84.

[47] Schreyer, D.J . and Skene, J .H.P. , Fate of GAP-43 in ascendingsp i n a l ax o ns o f D RG n eu ro n s a f t e r p e r i p h e ra l n e rv e i nj ury :d e l ay ed accu mu l a t i o n an d co r re l a t i o n w i t h reg en e ra t i v e p o t en -tial , J. Neurosci., 11 (1991) 3738-3751.[48] Skene, J .H.P. and Wil lard , M., Changes in axonal ly t ransportedprote ins during axon regenerat ion in toad re t inal gangl ion cel l s ,J. Cell Biol., 89 (1981) 86-96.

[49] Skene, J .H.P. and Wil lard , M., Characteri s t ics of growth-associ -a t ed p o l y p ep t i d e s i n reg en e ra t i n g t o ad re t i n a l g an g l i o n ce l laxons, J. Neurosci., 1 (1981) 419-426.[50] Skene, J .H.P. and WiUard , M., Axonal ly t ransp orted pro te insassocia ted wi th growth in rabbi t cent ra l and peripheral nervoussystem, Z Cell Biol., 89 (1981) 96-103 .

[5 1 ] Sk en e , J .H .P , G ro w t h -a sso c i a t ed p ro t e i n s an d t h e cu r i o u s d i -ch o t o mes o f n e rv e reg en e ra t i o n , Cell, 37 (1984) 697-700.

[52] Skene, J .H.P. , Jacob son, R .D., Snipes, G.J . , Mc Guir e , C.B. ,N o rd en , J . J . an d Freeman , J .A . , A p ro t e i n i n d u ced d u r i n g n e rv eg ro w t h (G A P-4 3 ) i s a ma j o r co mp o n en t o f g ro w t h co n e mem-b ran es , Science, 233 (1986) 783-786.

[53] Skene, J.H.P. , A x o n a l g ro w t h -a sso c i a t ed p ro t e i n s . Annu. Reu.Neurosci., 12 (1989) 127-156.

[5 4] Sk en e , J .H .P . an d V i rag , I . , Po s t - tran s l a t io n a l m emb ran e a t t a ch -men t an d d y n ami c fa t t y acy l a t i o n o f a n eu ro n a l g ro w t h co n ep ro t e i n , G A P-4 3 , J . Cell Biol., 108 (1989) 613-625.[5 5] So mm erv a i l le , T ., R ey n o l d s , M.L . an d W o o l f , C . J ., T i m e-d e -p en d en t d i f fe ren ces i n t h e i n c rea se i n G A P-4 3 ex p re ss i o n i nd o rsa l ro o t g an g l i o n ce l l s a f t e r p e r i p h e ra l ax o t o my , Neu ro -science, 45 (1991) 213-220.[56] Tetz laff , W ., Zwiers , H. , L ederis , K. , Cassar, L. and Bisby,M.A . , A x o n a l t ran sp o r t an d l o ca l i za t i o n o f B-5 0 / G A P-4 3 -1 i k ei mmu n o reac t i v i t y in reg en e ra t i n g sc i a t i c an d fac i a l n e rv es o f t h erat , Z Neurosci., 9 (1989) 1303-1313.

[57] Tetz laff , W., Alexa nder, S.W., M i l ler , F.D. and Bisby, M.A .,Resp o n se o f fac i a l an d ru b ro sp i n a l n eu ro n s t o ax o t o my : ch an g esi n mRN A ex p re ss i o n fo r cy t o sk e l e t a l p ro t e i n s an d G A P-4 3 , J .Neurosci., 11 (1991) 2528-2544.

[5 8 ] V an Lo o k e ren Camp ag n e , M. , O es t re i ch e r , A .B . , V an Be rg enEn H en eg o u w en , P .M.P . an d G i sp en , W .H . , U l t ra s t ru c t u ra li mmu n o cy t o ch emi ca l l o ca l i za t i o n o f B-5 0 / G A P-4 3 , a p ro t e i nkinase C subst ra te , in i so la ted presynapt ic nerve terminals andn eu ro n a l g ro w t h co n es , J. Neurocytol., 18 (1989) 479-489.[5 9 ] V an Lo o k e ren Camp ag n e , M. , O es t re i ch e r , A .B . , V an Be rg enEn H en eg o u w en , P .M.P . an d G i sp en , W .H . , U l t ra s t ru c t u ra ld o u b l e l o ca l i za ti o n o f B-5 0 / G A P4 3 an d sy n ap t o p h y s in (p . 3 8 ) int h e n eo n a t a l an d ad u l t ra t h i p p o camp u s , J. Neurocytol., 19(1990) 948-961.

[6 0] V an L o o k e ren C amp ag n e , M. , O es t re i ch e r , A .B . , Bu ma , P . ,V e rk l e i j , A . J , an d G i sp en , W .H . , U l t ra s t ru c t u ra l l o ca l i za t i o n o fad ren o co r t i co t ro p h i c h o rmo n e an d t h e p h o sp h o p ro t e i n B-5 0 / G ro w t h -A sso c i a t ed Pro t e i n 4 3 i n f reeze -su b s t i t u t ed , l o w i c ry lH M2 0 -en b ed d ed mesen cep h a l i c cen t ra l g ray su b s t an ce o f t h erat , Neuroscience, 42 (1991) 517-529.[6 1 ] V an Lo o k e ren Camp ag n e , M. , D o t t i , C ,G . , V e rk l e i j , A . J . , G i s -p en , W .H . an d O es t re i ch e r , A .B . , B -5 0 / G A P-4 3 l o ca l i za ti o n o nmem b ran es o f p u t a t i v e t ran sp o r t v e s i c l e s i n t h e ce l l b o d y , n eu -r i t e s an d g ro w t h co n es o f cu l t u re s h i p p o camp a l n eu ro n s , N e u -rosci Let t . , 137 (1992) 129-132.[6 2] V an Lo o k e ren Cam p ag n e , M . , D o t t i , C .G . , J ap T j o en San ,E .R .A . , V e rk l e i j , A . J . , G i sp en , W .H . an d O es t re i ch e r , A .B . ,B-5 0 / G A P-4 3 l o ca l i za t i o n i n p o l a r i zed h i p p o camp a l n eu ro n s i nvi t ro : an u l t rast ructural quant i ta t ive s tudy, Neuroscience, 50(1992) 35-52.

[6 3 ] V an d e r Zee , C .E .E .M. , N i e l an d e r , H .B . , V o s , J .P . , Lo p es d aSi lva , S. , Ver haag en, J . , Oest re ich er, A. , Schrama , L.H., Scho t -man , P . an d G i sp en , W .H . , Ex p re ss i o n o f g ro w t h -a sso c i a t edp ro t e i n B-5 0 (G A P-4 3 ) i n d o rsa l ro o t g an g l i a an d sc i a t i c n e rv ed u r i n g reg en e ra t i v e sp ro u t i n g , J . Neurosci., 9 (1989) 3505-3512.

[64] Ver ge, V.M.K., Tetz laff , W., Richar dson, P .M. and Bisby , M.A.,Co r re l a t i o n b e t w een G A P-4 3 an d n e rv e g ro w t h fac t o r recep t o rsin ra t sensory neurons, J . Neurosci., 10 (1990) 926 -936.

[6 5 ] V e rh aag en , J . , O es t re i ch e r , A .B . , Ed w ard s , P .M. , V e l d man , H . ,Jen n ek en s , F .G . I . an d G i sp en , W .H . , L i g h t - an d e l ec t ro n -mi c ro -sco p i ca l s t u d y o f p h o sp h o p ro t e i n B-5 0 fo l lo w i n g d en e rv a t i o nan d re i n n e rv a t i o n o f t h e ra t so l eu s mu sc l e , J. Neurosci., 8 (1988)1759-1766.[66] Woolf, C.J . , Reynolds, M.L. , Chong, M.S. , Emson, P. , Irwin , N.an d Ben o w i t z , L .I . , D en e rv a t i o n o f t h e mo t o r en d p l a t e re su l t s i nt h e rap i d ex p re ss io n b y t e rmi n a l Sch w an n ce l l s o f t h e g ro w t h -a s -so c i a t ed p ro t e i n G A P-4 3 , J . Neurosci., 12 (1992) 3999-401 0.

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