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Cytochalasin B and the intra-axonal movement of noradrenaline storage vesicles
Brahl Research, 49 (1973) 417-421 © Elsewer Scientific Pubhshmg Company, Amsterdam - Printed in The Netherlands
417
Cytochalasin B and the intra-axonal movement of noradrenaline storage vesicles
P BANKS, D. MAYOR AND P. MRAZ* Department oJ Btochemtstry and Department of Human Biology and Anatomy, Sheffield Untver~lt), Sheffield SIO 2TN (Great Brttam)
(Accepted October 16th, 1972)
The movement of noradrenahne storage vesicles along the axons of noradrenerglc neurones from their site of synthesis in the perikaryon to their destination m the varlcosities of the terminal axonal network, is generated locally within the axonst,L This movement can be prevented by colchicine and vinblastlne which cause the axonal mlcrotubules to disappear 2,a& Thus the movement of noradrenahne storage vestcles appears to depend upon the integrity of the axonal system of mlcrotubules. The nature of this dependence is unknown; the vesicles and tubules may undergo some kind of ATP-dependent interaction 9 or the tubules may merely ensure that the overall structure of the axon permits some unknown motile system to operate. In order to discover more about the mechanisms involved in intra-axonal transport ~t is useful to examine the effects of agents which appear to be specific inhlbltors of different kinds of cellular motility. Colchicine and vmblastine are agents of this type and have provided useful information about axonal transport Another interesting agent is cytochalasin B which inhibits a wide range of motile processes in cells, including cytoklnesls, cytoplasmic streaming, axonal elongation, noradrenahne and histamine release and platelet induced contraction of blood clots 7,1° 12. It has been claimed that cytochalasln B disrupts an lntracellular system of mlcrofilaments which is revolved in these dwerse phenomena. This view has met with fairly widespread acceptance, although Carter 4 considers that this generalization is not sufficiently well founded to allow the statement that 'sensitivity to the drug implies presence of some type of contractile mlcrofilament system '11 to be accepted uncritically Although the nature of the system inhibited by cytochalasm B is not known with certainty there is no doubt that it is an inhibitor of some kmds of cell motility and not of others such as karyogenesls and sperm tad and clhary function 11 Hence cytochalasm B can be used as a tool to delineate and classify different types of cellular motility This paper examrues the question of whether or not the colchicine and vinblastme sensitive movement of noradrenaline storage vesicles is also inhibited by cytochalasin B. * BrRlsh Council Scholar on leave of absence from Anatomtcky Ustav, Komensky University, Sasmkova 2, Bratlslava, Czechoslovakia.
41N
MI()RI
( ,,M",I{ N;~
,,
Tight silk hgaturcs ~ere apphed to the hypogastHc nerves ol adutl CalL, I ~'I,~ , the lnl'erloi mesemer~c ganglion and the hgated ner\e~ were remo~c,.l and lllCt[in,l~,d m a specmll 5 designed t w o - c o m p a i t m e n t box described in a prc~,,m, paper , ~,~_ ganglion and the proxmml part of the two nerve trunks were placed m one comp ~, ment and the more &sial. llgated, pall ol the ner,~e t r u n k s passed thlough a allI~.c!~c grease seal mto the other c o m p a r t m e n t Both c o m p a r t m e n t s c o n t a m e d E a g l e , MI M tissue culture m e d m m gassed ~xlth 95"o 02 5",, CO., and supplen|ented ~',.~h penicllhn (100 U/ml), streptomycin (100/tg/ml) a n d nystatin (25 U roll The s,_~lccl box was m a i n t a i n e d at 37 ( in a water bath for 24 h U s i n g t h t s apparatub, cxi,> chalasin B could be present in the medlun3 bathing the distal, hgated, portions oi l i~e nerve t r u n k ~hllst being denied act.es,, to the ganglion Thus effect.., of the d r t ~ ,.n axonal transport could bc ~solated l i o m an) effects ~t may haxc on Ihe n e u l o n a l perlkaryon The /'allure oJ [11C]acetat e added to the medium m the nerxc trtmk c o m p a r t m e n t to enter the g a n g h o n c o m p a r t m e n t indicated that there ~sas no leakage between the two c o m p a r t m e n t s Cytochalasm B (ICI L i d , U K ) ~as d l s s o b e d m dlmethylsulphox|de to gixe a stock solution of I mg/ml. For expefHnents mw31~,ng cytochalasin B, 0 2 or 0.4 ml samples of stock soluhon were d~luted to 40 ml v~tl3 Eagle's m e d m m to give hnal cytochalasm concentrations of 5 /;g/ml and 10 !;g. ml respectively. At the end o! the experiment the n o r a d r e n a h n e content ol the 2.5 mm ,31 length of nerve t r u n k ~mmediateiy prox|mal to the ligature ~sas measured ltuor~metrically as described previously"-' For electron m~croscopy the 1 mm piece o[ nerxe immediately proximal to the site of constrict|on and small pieces of nerxe from a region halfway between the constrmtion a n d the grease barrier ~ e r e fixed in 5'~,, glutaraldehyde in 0 [ M phosphate buffer (pH 7.3) at room temperature (approxim a t e l y 2 0 ' C ) f o r 2 5 h After washing for 24-48 h in 0.1 M phosphate buffer with 10"~,, sucrose, the tissue was post-osmicated in o s m m m tetroxide in Millomg's p h o s p h a t e buffer, dehydrated m graded ethanols, passed t h r o u g h epoxy propane and e m b e d d e d m Araldite UItrathln sections were stained with I o a q u e o u s urany[ acetate followed by lead citrate s and examined m either a Phihps 200 or an AEI 6B electron m~closcope The n u m b e r s of mlcrotubules per axon were counted m r a n d o m field, of transverse sections of the nerves halfway' between the constriction and the grease barrier As indicated in Table | the presence of cytochalasin B in the nerve t r u n k corn-
Figs 1 and 2. Electron micrographs taken from a point approximated 0 5 mm Immediately proMmal to the site of the constriction after incubation for 24 h m the presence of cytoehalasm B (5/~g'ml) Magmficatmn markers represent I /tin Fig 1 The accumulation of noradrenahne btorage dense-cored vesicles (d), mitochondrla (mL and myehn figures tmf) m swollen and distorted non-myehnated axons (A) Is illustrated. These appearances were ~denncal with those seen m control mcubatmns F~g 2 The double membranous complexes (arlowed) seen m some Schwann cells (S) Microtubules (t) are wstble m the transverse sections of the non-myehnated axons of more normal dmlenslon~ (A 1 Neurofilaments (f) can be seen in axon A1 and in the myehnated axon Profiles A2 and A3 are patt~ of swollen non-myehnated axon~ exhlbmng organelle accumulations s~mllar to those seen m Fig I
420 TABLE
SItORI t ~.)MMI IND. :~ ! ~ ~'.'b
I
LA(K OF I~[Fb-C[ OF ( Ylt)( fl~k[ XSIN B I)N NORADRkNAI. INI~ &t(UMULAFION A(~AINSI ~ LI(~AllJR[ A g a n g h o n / n e r v e p r e p a l a t ~ o n 'AdS i n c u b a t e d m E a g l e ' s l~ssue c u l t u r e m e d m m m a ~ v ~ o - c o m p a r t m ~ } t b o x a t 37 C f o r 24 h a s d e s c r i b e d m t h e t e x t C y t o c h a l a s m B w a s p r e s e n t m t h e n e r v e t ~ u n k c o m p a t tm e n t m all e x c e p t t h e c o n t r o l e x p e r i m e n t s T h e g a n g h o n c o m p a r t m e n t a l w a y s ~ o n t a m e d E a g l e s m e d m m w ~ t h o u t c y t o c h a l a s m B T h e n o r a d r e n a h n e c o n t e n t o f 2 5 m m o f n e r v e p r o x i m a l t o t h e ct,n str~ctlon a t z e r o t~me w a s a b o u t ( / 0 0 7 n m o l e ~ / n e r v e " T h e f i g u r e s m p a r e n t h e s e s i n d i c a t e t h e n u m b e r of observatlons
Dt"llg It11169l,t, It/lllk COttlpal /lilt,H/
Norad~ e n a h n e a~ Clglllldbl[lOIl 1'~ o ~m u d to hgature (tllllo[6"s lVA:nerve [ S t2 ~ l )
None C y t o c h a l a s m B (5/~g,,'ml~ Cytochalasm B (10/~g/ml) Colchlcme ( 10/¢g/ml)
017 I 002 0 19 0 02 0 18 0 03 0 10 ~ 0 0 2
(8) (5~ (2) 14~
partment had no effect upon the accumulation of noradrenahne against the constrictions at concentrations of 5 #g/ml and l0 t~g/ml during 24 h periods of incubation. In control experiments noradrenatine storage (dense-cored) vesmles accumulated proximal to the site of constriction in swollen and distorted non-myelinated axons together with other organelles, in particular mitochondna, agranular vesicles, dense bodies (myehn-figures) and tubulo-vesmular profiles of axonal endoplasmlc reticulum After incubation with cytochalasin B (5 #g/ml) there was an identical accumulation of dense-cored vesicles, mltochondria and other organelles (Fig. 1). Furthermore this drug had no effect on the abundance of microtubules seen in transverse section of the axons several mdhmeters proximal to the constriction. In control incubations the number of mmrotubules/axon was 41.6 ._ 2.1 S.E.M. (30 axons) and after lncubaUon with cytochalasin B (5 #g/ml) there were 38.3 _] 2.0 S E.M. (30 axons). The presence of colchicme (t0 #g/ml) in the nerve trunk compartment caused a 41% reduction m noradrenaline accumulation (see Table i) and the number of microtubules present per axon fell to 7 4 0.8 S.E M. (50 axons), in the present experiments, in whlch fixatmn was carried out at room temperature, the number of microtubules present per control axon was larger (41,6 : 2 I S.E.M.) than that found in previous experiments (16.5 ~ 0.5 S.E M.) when the tissue was fixed at 0 :C z,a. This is probably due to the fact that mlcrotubules are more stable at room temperature than at 0 C After treatment with cytochalasin B (5 pg/ml)many Schwann cells investing the non-myehnated axons and several axons developed a complex double-membrane formahon ( Fig 2). These were not seen either in control experiments or after incubation with DMSO alone. The precise origin and nature of these membranous formations is at present unknown but they appeared to have been induced by the presence of cytochalasin B. The present experiments indicate that cytochalasin B does not inhlbit the prommo-distal movement ofnoradrenahne storage (dense-cored) vesicles within the non-
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421
m y e h n a t e d a x o n s o f n o r a d r e n e r g ~ c n e u r o n e s . It w o u l d a p p e a r , t h e r e f o r e , t h a t t h e m o t i l e m e c h a n i s m s r e v o l v e d in this p r o c e s s differ f r o m t h o s e m v o l w n g a c y t o c h a l a s m s e n s i t i v e p r o c e s s s u c h as c y t o k i n e s l s a n d t h e e x t e n s i o n o f a x o n a l g r o w t h cones. If, as was s u g g e s t e d by W e s s e l l s et al. H, c y t o c h a l a s i n B i n t e r a c t s w i t h a n d m o d i f i e s a s y s t e m o f m~crofilaments, it w o u l d a p p e a r t h a t s u c h a s y s t e m c a n n o t
be i n v o l v e d in t h e
m ~ g r a t l o n o f n o r a d r e n a l l n e s t o r a g e vesicles. T h e r e was no e v i d e n c e to suggest t h a t this d r u g h a d a n y m a r k e d effect o n t h e o c c u r r e n c e o r t h e m o r p h o l o g y o f a x o n a l n e u r o filaments. F u r t h e r m o r e , u n l i k e c o l c h i c i n e , c y t o c h a l a s m B did n o t c a u s e t h e d i s p e r s i o n o f i n t r a - a x o n a l m i c r o t u b u l e s w h i c h a r e b e h e v e d to be r e v o l v e d m t h e m o v e m e n t o f d e n s e - c o r e d vesicles. F i n a n c i a l s u p p o r t f o r thls w o r k f r o m t h e W e l l c o m e T r u s t , t h e S m i t h K h n e a n d French Foundation,
the M . R . C
a n d S . R . C . is a c k n o w l e d g e d w i t h g r a t i t u d e . W e
t h a n k D r . C. W. P o t t e r for s u p p l i e s o f E a g l e ' s M e d i u m P . M . a c k n o w l e d g e s w i t h t h a n k s the r e c e i p t o f a British Counc~l S c h o l a r s h i p
I BANKS, P , MANGNALL, D. AND MAYOR, D , The re-distribution of cytochrome oxldase, nor-
adrenaline and adenosine trlphosphate In adrenerglc nerves constricted at two points, J Physlol (Lond), 200 (1969) 745-762 2 BANKS, P , MAYOR, D., MITCHELL, M., AND TOMEINSON, D , Studies on the translocatlon of noradrenahne storage vesicles In postganghonlc sympathetic neurones tn i,ttro. I n h b m o n of movement by colchlclne and vlnblastlne and evidence for the Involvement of axonal microtubules, J Phystol (Lond), 216 (1971) 625-639 BANKS, P., M&.YOR, O., AND TOMLINSON, D , Further evidence for the involvement of microtubules m the lntra-axonal movement of noradrenallne storage granules, J Physiol (Land), 219 (1971) 755 761. 4 CARVER, S B., The cytochalaslns as research tools tn cytology, Endeai'our, 31 (1972) 73 82 5 DAHLSTROM, A , AND HAGGENDAL, J., Studies on the transport and life-span of amine storage granules m a peripheral adrenerglc neuron system, Acta phy~tal scand, 67 (1966) 278 288 6 HOKFELT, T , AND DAHLSTROM,A , Electron microscopic observations on the distribution and transport of noradrenahne storage particles after local treatment with mltosIs mhlbltors, mcta phystol ~cand,Suppl 357 (1971) 10-11. 7 0 R R , T S. C , HALL, D. E , AND ALLISON, A. C., The role of contractile m~crofilaments in the release of histamine from mast cells, Nature (Lond.), 236 (1972) 350 351. 8 REYNOLDS, E S , The use of lead citrate at high pH as an electron-opaque stare m electron mlcroscop), J Cell Blol, 17 (1963) 208 212 9 SCHMITT, F O , Fibrous proteins and neuronal dynamics In S H BARONDES(Ed), Cellular D.vnamicv of the Neuron, Syrup of the Int Soc for Cell Biology, Vol 8, Academic Press, New York, 1969 10 THOA, N B, WOOTEN, G F , AnELROD, J , AND KOPIN, I J , lnhibmon of release of dopamme/3-hydroxylase and norepmephrine from sympathetic nerves by colchlclne, vlnblastlne and cytochalasm B, Proc nat Acad. Scl ( W a s h ) , 69 (1972) 520-522 11 WESSELLS, N , SPOONER, B S , ASH, J F , BRADLEY, M O , LUDUENA, M A , TAYLOR, E L , WRENN, J J., AND YAMADA, K. M , MIerofilaments m cellular and developmental processe~, Sctence, 171 (1971)135-143 12 YAMADA,K M , SPOONER, B S , AND WESSELLS, N K , Axonal growth roles of mtcrofilaments and mlcrotubules, Proc nat Acad. S c t . ( Wash ), 66 (1970) 1206
417
Cytochalasin B and the intra-axonal movement of noradrenaline storage vesicles
P BANKS, D. MAYOR AND P. MRAZ* Department oJ Btochemtstry and Department of Human Biology and Anatomy, Sheffield Untver~lt), Sheffield SIO 2TN (Great Brttam)
(Accepted October 16th, 1972)
The movement of noradrenahne storage vesicles along the axons of noradrenerglc neurones from their site of synthesis in the perikaryon to their destination m the varlcosities of the terminal axonal network, is generated locally within the axonst,L This movement can be prevented by colchicine and vinblastlne which cause the axonal mlcrotubules to disappear 2,a& Thus the movement of noradrenahne storage vestcles appears to depend upon the integrity of the axonal system of mlcrotubules. The nature of this dependence is unknown; the vesicles and tubules may undergo some kind of ATP-dependent interaction 9 or the tubules may merely ensure that the overall structure of the axon permits some unknown motile system to operate. In order to discover more about the mechanisms involved in intra-axonal transport ~t is useful to examine the effects of agents which appear to be specific inhlbltors of different kinds of cellular motility. Colchicine and vmblastine are agents of this type and have provided useful information about axonal transport Another interesting agent is cytochalasin B which inhibits a wide range of motile processes in cells, including cytoklnesls, cytoplasmic streaming, axonal elongation, noradrenahne and histamine release and platelet induced contraction of blood clots 7,1° 12. It has been claimed that cytochalasln B disrupts an lntracellular system of mlcrofilaments which is revolved in these dwerse phenomena. This view has met with fairly widespread acceptance, although Carter 4 considers that this generalization is not sufficiently well founded to allow the statement that 'sensitivity to the drug implies presence of some type of contractile mlcrofilament system '11 to be accepted uncritically Although the nature of the system inhibited by cytochalasm B is not known with certainty there is no doubt that it is an inhibitor of some kmds of cell motility and not of others such as karyogenesls and sperm tad and clhary function 11 Hence cytochalasm B can be used as a tool to delineate and classify different types of cellular motility This paper examrues the question of whether or not the colchicine and vinblastme sensitive movement of noradrenaline storage vesicles is also inhibited by cytochalasin B. * BrRlsh Council Scholar on leave of absence from Anatomtcky Ustav, Komensky University, Sasmkova 2, Bratlslava, Czechoslovakia.
41N
MI()RI
( ,,M",I{ N;~
,,
Tight silk hgaturcs ~ere apphed to the hypogastHc nerves ol adutl CalL, I ~'I,~ , the lnl'erloi mesemer~c ganglion and the hgated ner\e~ were remo~c,.l and lllCt[in,l~,d m a specmll 5 designed t w o - c o m p a i t m e n t box described in a prc~,,m, paper , ~,~_ ganglion and the proxmml part of the two nerve trunks were placed m one comp ~, ment and the more &sial. llgated, pall ol the ner,~e t r u n k s passed thlough a allI~.c!~c grease seal mto the other c o m p a r t m e n t Both c o m p a r t m e n t s c o n t a m e d E a g l e , MI M tissue culture m e d m m gassed ~xlth 95"o 02 5",, CO., and supplen|ented ~',.~h penicllhn (100 U/ml), streptomycin (100/tg/ml) a n d nystatin (25 U roll The s,_~lccl box was m a i n t a i n e d at 37 ( in a water bath for 24 h U s i n g t h t s apparatub, cxi,> chalasin B could be present in the medlun3 bathing the distal, hgated, portions oi l i~e nerve t r u n k ~hllst being denied act.es,, to the ganglion Thus effect.., of the d r t ~ ,.n axonal transport could bc ~solated l i o m an) effects ~t may haxc on Ihe n e u l o n a l perlkaryon The /'allure oJ [11C]acetat e added to the medium m the nerxc trtmk c o m p a r t m e n t to enter the g a n g h o n c o m p a r t m e n t indicated that there ~sas no leakage between the two c o m p a r t m e n t s Cytochalasm B (ICI L i d , U K ) ~as d l s s o b e d m dlmethylsulphox|de to gixe a stock solution of I mg/ml. For expefHnents mw31~,ng cytochalasin B, 0 2 or 0.4 ml samples of stock soluhon were d~luted to 40 ml v~tl3 Eagle's m e d m m to give hnal cytochalasm concentrations of 5 /;g/ml and 10 !;g. ml respectively. At the end o! the experiment the n o r a d r e n a h n e content ol the 2.5 mm ,31 length of nerve t r u n k ~mmediateiy prox|mal to the ligature ~sas measured ltuor~metrically as described previously"-' For electron m~croscopy the 1 mm piece o[ nerxe immediately proximal to the site of constrict|on and small pieces of nerxe from a region halfway between the constrmtion a n d the grease barrier ~ e r e fixed in 5'~,, glutaraldehyde in 0 [ M phosphate buffer (pH 7.3) at room temperature (approxim a t e l y 2 0 ' C ) f o r 2 5 h After washing for 24-48 h in 0.1 M phosphate buffer with 10"~,, sucrose, the tissue was post-osmicated in o s m m m tetroxide in Millomg's p h o s p h a t e buffer, dehydrated m graded ethanols, passed t h r o u g h epoxy propane and e m b e d d e d m Araldite UItrathln sections were stained with I o a q u e o u s urany[ acetate followed by lead citrate s and examined m either a Phihps 200 or an AEI 6B electron m~closcope The n u m b e r s of mlcrotubules per axon were counted m r a n d o m field, of transverse sections of the nerves halfway' between the constriction and the grease barrier As indicated in Table | the presence of cytochalasin B in the nerve t r u n k corn-
Figs 1 and 2. Electron micrographs taken from a point approximated 0 5 mm Immediately proMmal to the site of the constriction after incubation for 24 h m the presence of cytoehalasm B (5/~g'ml) Magmficatmn markers represent I /tin Fig 1 The accumulation of noradrenahne btorage dense-cored vesicles (d), mitochondrla (mL and myehn figures tmf) m swollen and distorted non-myehnated axons (A) Is illustrated. These appearances were ~denncal with those seen m control mcubatmns F~g 2 The double membranous complexes (arlowed) seen m some Schwann cells (S) Microtubules (t) are wstble m the transverse sections of the non-myehnated axons of more normal dmlenslon~ (A 1 Neurofilaments (f) can be seen in axon A1 and in the myehnated axon Profiles A2 and A3 are patt~ of swollen non-myehnated axon~ exhlbmng organelle accumulations s~mllar to those seen m Fig I
420 TABLE
SItORI t ~.)MMI IND. :~ ! ~ ~'.'b
I
LA(K OF I~[Fb-C[ OF ( Ylt)( fl~k[ XSIN B I)N NORADRkNAI. INI~ &t(UMULAFION A(~AINSI ~ LI(~AllJR[ A g a n g h o n / n e r v e p r e p a l a t ~ o n 'AdS i n c u b a t e d m E a g l e ' s l~ssue c u l t u r e m e d m m m a ~ v ~ o - c o m p a r t m ~ } t b o x a t 37 C f o r 24 h a s d e s c r i b e d m t h e t e x t C y t o c h a l a s m B w a s p r e s e n t m t h e n e r v e t ~ u n k c o m p a t tm e n t m all e x c e p t t h e c o n t r o l e x p e r i m e n t s T h e g a n g h o n c o m p a r t m e n t a l w a y s ~ o n t a m e d E a g l e s m e d m m w ~ t h o u t c y t o c h a l a s m B T h e n o r a d r e n a h n e c o n t e n t o f 2 5 m m o f n e r v e p r o x i m a l t o t h e ct,n str~ctlon a t z e r o t~me w a s a b o u t ( / 0 0 7 n m o l e ~ / n e r v e " T h e f i g u r e s m p a r e n t h e s e s i n d i c a t e t h e n u m b e r of observatlons
Dt"llg It11169l,t, It/lllk COttlpal /lilt,H/
Norad~ e n a h n e a~ Clglllldbl[lOIl 1'~ o ~m u d to hgature (tllllo[6"s lVA:nerve [ S t2 ~ l )
None C y t o c h a l a s m B (5/~g,,'ml~ Cytochalasm B (10/~g/ml) Colchlcme ( 10/¢g/ml)
017 I 002 0 19 0 02 0 18 0 03 0 10 ~ 0 0 2
(8) (5~ (2) 14~
partment had no effect upon the accumulation of noradrenahne against the constrictions at concentrations of 5 #g/ml and l0 t~g/ml during 24 h periods of incubation. In control experiments noradrenatine storage (dense-cored) vesmles accumulated proximal to the site of constriction in swollen and distorted non-myelinated axons together with other organelles, in particular mitochondna, agranular vesicles, dense bodies (myehn-figures) and tubulo-vesmular profiles of axonal endoplasmlc reticulum After incubation with cytochalasin B (5 #g/ml) there was an identical accumulation of dense-cored vesicles, mltochondria and other organelles (Fig. 1). Furthermore this drug had no effect on the abundance of microtubules seen in transverse section of the axons several mdhmeters proximal to the constriction. In control incubations the number of mmrotubules/axon was 41.6 ._ 2.1 S.E.M. (30 axons) and after lncubaUon with cytochalasin B (5 #g/ml) there were 38.3 _] 2.0 S E.M. (30 axons). The presence of colchicme (t0 #g/ml) in the nerve trunk compartment caused a 41% reduction m noradrenaline accumulation (see Table i) and the number of microtubules present per axon fell to 7 4 0.8 S.E M. (50 axons), in the present experiments, in whlch fixatmn was carried out at room temperature, the number of microtubules present per control axon was larger (41,6 : 2 I S.E.M.) than that found in previous experiments (16.5 ~ 0.5 S.E M.) when the tissue was fixed at 0 :C z,a. This is probably due to the fact that mlcrotubules are more stable at room temperature than at 0 C After treatment with cytochalasin B (5 pg/ml)many Schwann cells investing the non-myehnated axons and several axons developed a complex double-membrane formahon ( Fig 2). These were not seen either in control experiments or after incubation with DMSO alone. The precise origin and nature of these membranous formations is at present unknown but they appeared to have been induced by the presence of cytochalasin B. The present experiments indicate that cytochalasin B does not inhlbit the prommo-distal movement ofnoradrenahne storage (dense-cored) vesicles within the non-
SHORT COMMUNICATIONS
421
m y e h n a t e d a x o n s o f n o r a d r e n e r g ~ c n e u r o n e s . It w o u l d a p p e a r , t h e r e f o r e , t h a t t h e m o t i l e m e c h a n i s m s r e v o l v e d in this p r o c e s s differ f r o m t h o s e m v o l w n g a c y t o c h a l a s m s e n s i t i v e p r o c e s s s u c h as c y t o k i n e s l s a n d t h e e x t e n s i o n o f a x o n a l g r o w t h cones. If, as was s u g g e s t e d by W e s s e l l s et al. H, c y t o c h a l a s i n B i n t e r a c t s w i t h a n d m o d i f i e s a s y s t e m o f m~crofilaments, it w o u l d a p p e a r t h a t s u c h a s y s t e m c a n n o t
be i n v o l v e d in t h e
m ~ g r a t l o n o f n o r a d r e n a l l n e s t o r a g e vesicles. T h e r e was no e v i d e n c e to suggest t h a t this d r u g h a d a n y m a r k e d effect o n t h e o c c u r r e n c e o r t h e m o r p h o l o g y o f a x o n a l n e u r o filaments. F u r t h e r m o r e , u n l i k e c o l c h i c i n e , c y t o c h a l a s m B did n o t c a u s e t h e d i s p e r s i o n o f i n t r a - a x o n a l m i c r o t u b u l e s w h i c h a r e b e h e v e d to be r e v o l v e d m t h e m o v e m e n t o f d e n s e - c o r e d vesicles. F i n a n c i a l s u p p o r t f o r thls w o r k f r o m t h e W e l l c o m e T r u s t , t h e S m i t h K h n e a n d French Foundation,
the M . R . C
a n d S . R . C . is a c k n o w l e d g e d w i t h g r a t i t u d e . W e
t h a n k D r . C. W. P o t t e r for s u p p l i e s o f E a g l e ' s M e d i u m P . M . a c k n o w l e d g e s w i t h t h a n k s the r e c e i p t o f a British Counc~l S c h o l a r s h i p
I BANKS, P , MANGNALL, D. AND MAYOR, D , The re-distribution of cytochrome oxldase, nor-
adrenaline and adenosine trlphosphate In adrenerglc nerves constricted at two points, J Physlol (Lond), 200 (1969) 745-762 2 BANKS, P , MAYOR, D., MITCHELL, M., AND TOMEINSON, D , Studies on the translocatlon of noradrenahne storage vesicles In postganghonlc sympathetic neurones tn i,ttro. I n h b m o n of movement by colchlclne and vlnblastlne and evidence for the Involvement of axonal microtubules, J Phystol (Lond), 216 (1971) 625-639 BANKS, P., M&.YOR, O., AND TOMLINSON, D , Further evidence for the involvement of microtubules m the lntra-axonal movement of noradrenallne storage granules, J Physiol (Land), 219 (1971) 755 761. 4 CARVER, S B., The cytochalaslns as research tools tn cytology, Endeai'our, 31 (1972) 73 82 5 DAHLSTROM, A , AND HAGGENDAL, J., Studies on the transport and life-span of amine storage granules m a peripheral adrenerglc neuron system, Acta phy~tal scand, 67 (1966) 278 288 6 HOKFELT, T , AND DAHLSTROM,A , Electron microscopic observations on the distribution and transport of noradrenahne storage particles after local treatment with mltosIs mhlbltors, mcta phystol ~cand,Suppl 357 (1971) 10-11. 7 0 R R , T S. C , HALL, D. E , AND ALLISON, A. C., The role of contractile m~crofilaments in the release of histamine from mast cells, Nature (Lond.), 236 (1972) 350 351. 8 REYNOLDS, E S , The use of lead citrate at high pH as an electron-opaque stare m electron mlcroscop), J Cell Blol, 17 (1963) 208 212 9 SCHMITT, F O , Fibrous proteins and neuronal dynamics In S H BARONDES(Ed), Cellular D.vnamicv of the Neuron, Syrup of the Int Soc for Cell Biology, Vol 8, Academic Press, New York, 1969 10 THOA, N B, WOOTEN, G F , AnELROD, J , AND KOPIN, I J , lnhibmon of release of dopamme/3-hydroxylase and norepmephrine from sympathetic nerves by colchlclne, vlnblastlne and cytochalasm B, Proc nat Acad. Scl ( W a s h ) , 69 (1972) 520-522 11 WESSELLS, N , SPOONER, B S , ASH, J F , BRADLEY, M O , LUDUENA, M A , TAYLOR, E L , WRENN, J J., AND YAMADA, K. M , MIerofilaments m cellular and developmental processe~, Sctence, 171 (1971)135-143 12 YAMADA,K M , SPOONER, B S , AND WESSELLS, N K , Axonal growth roles of mtcrofilaments and mlcrotubules, Proc nat Acad. S c t . ( Wash ), 66 (1970) 1206