Fast axonal transport of the vesicular acetylcholine transporter (VAChT) in cholinergic neurons in the rat sciatic nerve

NEUROCHEMISTRY International Neurochem[ Int[ 21 "0887# 346Ð356

Fast axonal transport of the vesicular acetylcholine transporter "VAChT# in cholinergic neurons in the rat sciatic nerve Jia!Yi Li\0 Anna Maria Dahlstrom\0 Louis B[ Hersh\1 Annica Dahlstrom0 0

Department of Anatomy and Cell Biolo`y\ University of Gotebor`\ Medicinare`atan 4\ S!302 89 Gotebor`\ Sweden Department of Biochemistry\ University of Kentucky\ Chandler Medical Center\ Lexin`ton\ KY 39425!9973\ U[S[A[

1

Received 5 September 0886^ accepted 19 October 0886

Abstract The sciatic nerve\ as a part of the peripheral nervous system "PNS#\ has been used to study axonal transport for decades[ It contains motor\ sensory as well as autonomic axons[ The present study has concentrated on the axonal transport of the synaptic vesicle acetylcholine transporter "VAChT#\ using the {{stop!~ow:nerve crush|| method[ After blocking fast axonal transport by means of a crush\ distinct accumulations of various synaptic vesicle proteins\ including VAChT\ and peptides developed during the _rst hour after crush!operation and marked increases were observed up to 7 h post!operative[ Semiquantitative analysis\ using cyto~uorimetric scanning "CFS# of immuno!incubated sections\ revealed a rapid rate of accumulation proximal to the crush\ and that the ratio between distal accumulations "organelles in retrograde transport# and proximal accumulations "organelles in anterograde transport# was about 39)[ Most synaptic vesicle proteins were colocalized in the axons proximal to the crush[ VAChT!immu! noreactive axons were also immunoreactive for choline acetyltransferase "ChAT#[ Autonomic axons with VAChT also contained VIP!LI[ The results demonstrate "0# that VAChT\ as well as other synaptic vesicle proteins\ is transported with fast axonal transport in motor axons as well as in autonomic post!ganglionic neurons in this nerve\ "1# VAChT colocalized in motor axons with SV1 as well as with synaptophysin\ indicating storage in the same axonal particle\ "2# in the autonomic postganglionic sympathetic cholinergic _bres\ VAChT colocalized with VIP\ but VIP!LI was present in rather large granular structures while VAChT!LI was present mostly as small granular elements\ "3# in motor as well as in autonomic axons ChAT!LI was present in VAChT!positive axons\ and "4# the ratio of recycling "retrogradely accumulated# VAChT!IR was about 39)\ in contrast to the recycling fraction of synaptophysin that was about 69)[ Þ 0887 Elsevier Science Ltd[ All rights reserved[ Key words] Vesicular acetylcholine transporter "VAChT#^ Synaptic vesicle proteins^ Choline acetyltransferase^ VIP^ Axonal transport^ Cyto~uorimetric scanning^ Immuno~uorescence^ Sciatic nerve^ Rat^ Confocal microscopy

Introduction Neurons consist of perikarya with long processes\ which can be subdivided into axons and dendrites according to their properties[ Proteins are synthesized in the cell body where the synthetic machinery is located and packed into di}erent organelles before being exported into the axon and eventually to the nerve terminals[ Endocytosed materials within terminals will be recycled to the cell body\ e[g[ for feed!back signalling[ Several di}erent approaches have been used to study this phenomenon including biochemical methods\ Video!DIC\ immuno! ~uorescence\ etc "Dahlstrom et al[\ 0878# for several years\ the techniques of stop!~ow "crush# and cyto! ~uorimetric scanning "CFS# have been successfully  To whom all correspondence should be addressed ðTel[] ¦35!20! 6622243^ Fax] ¦35!20!718589^ E!mail] jiayi[liÝanatcell[gu[seŁ[ 9086Ð9075:87 ,08[99 Þ 0887 Elsevier Science Ltd[ All rights reserved PII] S 9 0 8 6 Ð 9 0 7 5 " 8 6 # 9 9 0 1 1 Ð 7

employed in our laboratory for axonal transport studies in the rat peripheral nervous system "Larsson et al[\ 0873\ 0876^ Dahlstrom et al[\ 0878^ Dahlstrom and Li\ 0883#[ The advantages of this CFS!technique are that several di}erent antigens can be studied in a single nerve speci! men\ thereby reducing biological variations\ and sim! ultaneously allowing the possibility to perform quantitative and morphological analyses in the same tissue[ The _rst neurotransmitter related substances to be studied using the stop!~ow technique were investigated using histochemical methods[ Acetylcholine esterase "AChE^ Lubin|ska 0853^ Lubin|ska and Niemierko\ 0860# was found to accumulate not only proximally but also distally to a nerve crush\ suggesting anterograde as well as retrograde fast transport of this enzyme[ This pioneering work by Lubin|ska demonstrated not only the occurrence of a fast transport\ in addition to the slow

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transport advocated by Weiss and Hiscoe "0837# at that time\ but also the phenomenon of retrograde accumu! lations[ Noradrenaline "NA# in postganglionic sym! pathetic neurons in the sciatic nerve was studied using the Hillarp!Falck ~uorescence method "Dahlstrom\ 0854#[ Subsequently biochemical assays of the material accumu! lated relative to the crush were performed^ for NA and for acetylcholine "ACh# and the two cholinergic enzymes "for review\ see Dahlstrom\ 0872#[ The rate of accumulation of ACh and AChE above a crush was found to be fast\ indicating fast axonal trans! port\ whereas the accumulation of choline ace! tyltransferase "ChAT# was much slower\ but somewhat di}ering in accumulation rates between di}erent per! ipheral nerves[ Some problems were encountered when studying the transport of ACh\ because of the elusive nature of the substance\ subjected to a continuous syn! thesis and degradation in the axon\ rendering de_nite estimations problematic "see disc[ in Dahlstrom et al[\ 0871#[ This was especially noticed when we made an attempt to investigate if the ACh accumulated proximal to a crush was in a particulate fraction\ and\ if so\ in which fraction "Heilbronn and Dahlstrom\ unpublished#[ A large number of the peripheral ends of crushed cat sciatic nerves were collected and subjected to homo! genization and sucrose gradient centrifugation\ and the content of ACh in di}erent fractions was measured[ The use of AChE!inhibitors was avoided\ in order to focus on ACh protected inside a particle membrane "Dahlstrom et al[\ 0863#[ A large portion of the accumulated ACh in this experiment was found in the fractions equilibrating above 0[1 M and 0[7 M of sucrose\ indicating the presence of a particle heavier than the synaptic vesicles[ We con! tinued the studies on the transport particle for ACh using SV1\ the newly presented antibody against Torpedo syn! aptic vesicles\ "Dahlstrom et al[\ 0870^ Booj et al[\ 0875\ 0876#\ _rst claimed to be speci_c for cholinergic vesicles[ However\ it was later demonstrated that these antibodies also react with proteins in vesicles of non!cholinergic neurons\ and therefore we could use neither of them to speci_cally investigate further the ACh!storing organelle[ The aim of this study was to use the newly developed antibody against the vesicular ACh transporter "VAChT# in the study of the axonal cholinergic organelle[ Since this transporter is so far not found to react with non! cholinergic neurons\ but\ in contrast\ to be very speci_! cally expressed in only neurons of cholinergic nature "Gil! more et al[\ 0885# it should be an excellent tool to study axonal transport of the {{cholinergic organelle||[ Materials and methods Operations Adult SpragueÐDawley male rats "199Ð149 g# were used[ Under sodium pentobarbital anaesthesia "i[p[ 29 mg:kg

b[w[#\ the sciatic nerves bilaterally were double!crushed as described earlier "Dahlstrom et al[\ 0878#[ The rats were transcardially perfused post!operatively at 9\ 0\ 2 or 7 h\ respectively under deep anaesthesia\ with 3) paraformaldehyde in 9[0 M PBS "pH 6[3#\ the sciatic nerves were dissected and post_xed in the same _xative for 3 h and rinsed in 09) sucrose:PBS overnight[ In one group of animals the lumbar sympathetic gan! glia "L2ÐL5# were exposed via an anterior approach and removed under an operation microscope[ This was done in order to remove the sympathetic axons in the sciatic nerve\ some of which are cholinergic in the adult animal\ innervating the sweat glands[ Thus\ we had one group of rats whose sciatic nerves contained cholinergic motor as well as sympathetic _bres\ and one group of sciatic nerves only containing cholinergic motor axons[ All procedures were in accordance with the permission of the Animal Ethical Committee in Goteborg[

Immuno~uorescence The sciatic nerves from these two groups were frozen with compressed CO1\ sectioned longitudinally in a cryo! stat "09 mm#\ and placed on gelatine coated glass slides[ Indirect immuno~uorescence incubations were carried out "Li et al[\ 0883# using the antibodies listed in Table 0[ All antibody preparations used have been char! acterized by the groups that produced them[ Thus\ the immuno~uorescence studied most likely represents the true antigens[ However\ since cross reactivity with other macromolecules cannot be totally ruled out we refer to amounts of\ e[g[ VAChT like immunoreactivity "LI# in this study[ For single labelling\ the sections were preincubated with normal sera "0 ] 49# from the species of the secondary antibodies for 0 h[ Incubations with the primary anti! bodies were carried out overnight\ followed by incubation with biotinylated goat!anti!rabbit! or rabbit!anti!goat! IgG "0 ] 199# "Vector Lab\ Burlingame\ CA\ U[S[A[#[ Streptavidin conjugated with ~uorescein "FITC# was used for visualizing the labelling\ as described by Li et al[ "0884#[ The double stainings between goat anti!VAChT and other rabbit antibodies were performed as follows[ Brie! ~y\ normal donkey serum was used for preincubation\ followed by primary antibody against goat anti!VAChT with rabbit anti!synaptophysin\ !VIP\ or !ChAT\ etc[ After rinsing\ the sections were incubated with donkey anti!goat IgG conjugated with Texas Red and donkey anti!rabbit conjugated with FITC "Jackson ImmunoLab\ Grove\ U[S[A[#[ Control sections\ included in every incu! bation series\ were incubated with normal goat\ rabbit or donkey sera\ instead of the primary antisera\ followed by all subsequent incubations as described above[ These sections were always negative for immuno~uorescence[

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J[!Y[ Li et al[:Neurochem[ Int[ 21 "0887# 346Ð356 Table 0 Primary antibodies used in the present study Antibodies "Codes#

Produced in

Source

Working dilution

References

VAChT "AB 0467# ChAT Synaptophysin "G 85#

goat rabbit rabbit

Chemicon0 L[ Hersh1 R[ Jahn2

0 ] 4999 0 ] 399 0 ] 1999

Bajanini et al[\ 0883

SV1 "09H# VIP "V!2497# TH

mouse rabbit rabbit

R[ Kelly3 Sigma4 M[ Goldstein5

0 ] 799 0 ] 49 0 ] 0599

Jahn et al[\ 0874 Navone et al[\ 0875 Buckley and Kelly\ 0874 Goldstein\ 0861 Goldstein et al[\ 0861

0

Goat!anti!VAChT\ "Chemicon AB 0467#\ produced against a synthetic peptide of the C!terminal region[ Rabbit anti!ChAT " from L[ Hersch# from 2 rabbits\ produced against human placental enzyme[ 2 Rabbit anti!synaptophysin\ produced against puri_ed synaptophysin[ 3 Mouse monoclonal anti!SV1\ recognizing the 84 kD integral synaptic vesicle membrane glycoprotein SV1 3[ 4 Rabbit anti!VIP\ "Sigma\ V!2497# produced against synthetic VIP[ 5 Rabbit!anti!tyrosine hydroxylase "TH#\ raised against TH isolated from rat pheochromocytoma cells\ dilution 0 ] 799[ 1

Cyto~uorimetric scannin` The immunolabelled sections were _rst scanned in the equipment for CFS as described earlier "Larsson et al[\ 0876^ Dahlstrom et al[\ 0878^ Dahlstrom and Li\ 0883#[ The proper dilution of antibodies was determined by testing a good correlation between intensity of immu! no~uorescence and antibody dilution "Li\ 0884#[ Fur! thermore\ identical scanning sensitivities were used for all sections compared[ The reliability of this method was previously established by comparing CFS data with those obtained by enzymatic methods "Larsson\ 0874^ Dahlstrom et al[\ 0878#[ Although care has been taken during the preparation of the samples\ and during the scanning procedures\ the data obtained from the CFS are still semiquantitative\ the amounts of anterograde versus retrograde "recycling# axonal transport of each protein are represented as relative amounts of immuno! ~uorescences[ After CFS scanning\ the sections were then viewed and photographed\ using Kodak Tri!X _lm\ in the confocal laser scanning microscope "CLSM\ BioRad MRC 599# "Amos et al[\ 0876#[ From each nerve 4Ð6 sections were immunoincubated with a particular antibody and scanned[ The mean of these measurements were taken as the result from one nerve[ In total 3Ð5 nerves were processed for each time and group[ Mean2S[E[M[ are shown in Fig[ 4[ Results Distribution of VAChT in motor neurons VAChT!LI was present in very low amounts in many of the motor perikarya in the spinal cord "Fig[ 0A\ B#\ but

all motor perikarya were ChAT!immunoreactive with medium to weak intensity "Fig[ 1C#[ Many large boutons with strong VAChT!immuno~uorescence were seen around the periphery of the large motor perikarya "Fig[ 0A\ B#[ The motor end!plates in the gastrocnemic muscles were strongly VAChT!immunoreactive as well as positive for synaptophysin with a complete colocalization:overlap "Fig[ 0C#[ The end plates were also ChAT!immuno! reactive but with a medium ~uorescence intensity "Fig[ 1D#[ In intact myelinated large axons VAChT!LI could not be detected "Fig[ 0E# but proximal to a crush strongly ~uorescent accumulations were seen in large axons "arrows in Fig[ 0F\ G#[ These accumulations were also synaptophysin! and SV1!positive\ but the number of syn! aptophysin!containing axons with VAChT!accumu! lations were also ChAT!immunoreactive "Fig[ 1A\ B#[ The ChAT!LI appeared in many cases to trail ahead of the vesicle antigens\ especially noticed at the nodal areas "Fig[ 1E#[

Distribution of VAChT in lumbar autonomic neurons In the lumbar sympathetic ganglia a small number of cell bodies showed very weak VAChT!LI "Fig[ 2C#\ as well as weak to medium ChAT!LI "not shown#[ Nerve terminals basketing around the cell bodies were strongly VAChT! immunoreactive "Fig[ 2C#[ The few VAChT!positive cellbodies were not TH!immunoreactive\ in contrast to the majority of ganglion cells "Fig[ 3C\ D#\ but positive for VIP "Fig[ 2C#[ In the sciatic nerve with very weak VAChT!immuno! ~uorescence bundles of axons detected proximal to and close to the crush where accumulations in autonomic

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Fig[ 0[ Confocal laser scanning photographs of rat tissue sections doubly incubated with anti!synaptophysin "p27\ left lane# and anti!VAChT "right lane#[ "A and B# Anterior horn of the spinal cord lumbar intumescence[ Motor perikarya contain weakly ~uorescent granular VAChT!LI in a perinuclear zone\ and are surrounded by big nerve terminals _lled with strongly ~uorescent VAChT!LI "right lane#[ Synaptophysin "left lane# cannot be detected in the motor somata\ but is present in a large number of nerve terminals in the neuropil and around the motor perikarya[ Synaptophysin! immunoreactive nerve terminal boutons appear to be smaller than the big VAChT!immunoreactive terminals^ merging of the two channels indicated a partial colocalization[ Bars are 49 mm "A# and 14 mm "B#[ "C# Motor end!plate in the gastrocnemic muscle with a perfect colocalization between synaptophysin "left lane# and VAChT!LI "right lane#[ Bar is 14 mm[ "D# Section of the rat|s hind paw[ Tubular sweat glands and their vasulature contain many synaptophysin!positive nerve terminals\ some of which also contain VAChT!immunoreactive material[ Bar is 49 mm[ "E# From a sciatic nerve crush operated 9 h before sacri_ce[ Arrows indicate the crush area[ A few thin axons with synaptophysin!LI are present\ but VAChT! immunoreactivity is very low\ or absent[ Bar is 099 mm[ "F# Accumulations of synaptophysin!LI and VAChT!LI proximal to a 0 h crush "arrows#[ It is clear that all VAChT!positive axons also contain synaptophysin\ but the number of synaptophysin containing axons is much larger\ since synaptophysin is also present in other\ non!motor axons in the sciatic nerve[ Bar is 099 mm[ "G# Section of a sciatic nerve which was crush operated 7 hrs before sacri_ce[ Arrows indicate site of crush\ separating the segment of nerve proximal to the crush from the area distal to the crush[ It is very clear that VAChT accumulations\ present at a distance from the crush where myelinated motor axons accumulate\ occur only on a subset of synaptophysin containing axons "small arrows#[ Distal to the crush weakly immunoreactive accumulations can be seen[ The majority of the synaptophysin positive axons represent unmyelinated\ autonomic _bres\ which contain the post!crush accumulations close to the crush area\ proximal as well as distal to the lesion[ Bar is 099 mm[ "H# High magni_cation picture of accumulated motor axons proximal to a crush[ In two axons synaptophysin and VAChT are present\ with a similar intra!axonal distribution[ Two other axons contain synaptophysin "left lane# but no detectable VAChT[ Bar is 14 mm[

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Fig[ 1[ Confocal laser scanning photographs from rat tissue sections doubly incubated with anti!ChAT "left lane# and anti!VAChT "right lane#[ "A# Section of sciatic nerve crush operated "arrows# 0 h before sacri_ce[ The VAChT accumulations proximal to the crush also contain accumulations of ChAT!LI[ Distal to the crush\ some weakly ~uorescent ChAT!positive axons are present\ a few of which also contain VAChT!LI Bar is 099 mm[ "B# Section of sciatic nerve 7 h after crushing "arrows#[ The axons with accumulations of VAChT!LI are also ChAT!immunoreactive\ proximal as well as distal to the crush[ However\ it appears that there are more axons with ChAT!LI than VAChT!LI[ Bar is 099 mm[ "C# Two motor perikarya in the lumbar spinal cord\ with strong ChAT!immunoreactivity\ also seen in an axon "bottom right in the left lane#[ Both cell bodies also contain weakly immunoreactive VAChT!positive small granules\ as well as strong VAChT!~uorescence in nerve terminals around the cell bodies[ Bar is 14 mm[ "D# Two motor end!plates from the gastrocnemic muscle with strong VAChT!LI and weak\ but colocalized\ ChAT!immuno~uorescence[ Bar is 14 mm[ "E# A motor axon\ proximal to a crush\ contains ChAT!LI and colocalized VAChT!LI[ Distal to the Ranvier|s node immunoreactive material has passed distally\ but ChAT!immunoreactive material is seen to trail ahead of the VAChT!material[ Bar is 09 mm[

unmyelinated _bres are known to develop "cf[ Li et al[\ 0883\ 0885a#[ However\ it was clear that vesicles accumu! lating in these autonomic axons contained very low detectable amounts of the transporter protein\ in contrast to the motor axons "above#[ Some ChAT!LI could be seen in this area "Fig[ 1A\ B# and also VIP "Fig[ 2A#[ We collected sections of the paws\ and could observe rather few VAChT!positive terminals\ which colocalized well with synaptophysin or SV1 "Fig[ 0D#\ and also contained VIP!LI "arrows in Fig[ 2D#[ In lumbar sym!

pathectomized rats\ the TH!immunoreactive autonomic axons were totally abolished "Fig[ 3B#\ suggesting a suc! cessful lumbar sympathectomy\ but\ as noted in Fig[ 3\ there was no clear reduction in the number of VAChT! positive axons after sympathectomy\ indicating that the vast majority of axons with VAChT!accumulation were\ in fact\ motor axons[ However\ in the few VIP!containing axons that were also VAChT!positive\ the intra!axonal subcellular distribution clearly showed that VIP!immu! no~uorescence was present in large granular structures\

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Fig[ 2[ Confocal laser scanning photographs from rat tissue sections doubly incubated with anti!VIP "left lane# and anti!VAChT "right lane#[ "A# Section from a sciatic nerve which was crush!operated 7 h before sacri_ce[ Proximal to the crush "arrows# some axons contain accumulations of VIP! positive as well as VAChT!positive material "small arrows#[ Many axons are VAChT!positive\ but VIP negative\ and vice versa[ Distal to the crush\ some thin axons contain both VIP!LI and VAChT!LI[ Bar is 099 mm[ "B# Ventral horn of the lumbar spinal cord with some motor perikarya containing granular\ weakly ~uorescent VIP!LI[ These perikarya are surrounded by large VAChT!positive nerve terminals[ Bar is 49 mm[ "C# Section from lumbar sympathetic ganglia\ showing one VIP!containing nerve cellbody\ and a few VIP!positive axons[ A large number of nerve terminals surrounding the VIP!positive and VIP!negative perikarya are strongly VAChT!immunoreactive[ Bar is 49 mm[ "D# Section from the hind!paw of a normal animal\ showing sweat glands innervated by many strongly ~uorescent VAChT!positive nerve terminals\ some of which are also VIP!positive "small arrows#[ "E# Section from a hind!paw from a lumbar sympathectomized rat[ Number of nerve terminals with VIP!LI and VAChT!LI are reduced\ or abolished by the sympathectomy operation[ Bar is 49 mm[ "F# One axon from the proximal area of a 7 h crush!operated sciatic nerve[ VIP!LI is distributed in rather large granular structures\ while VAChT!LI is present in the same axon\ but with a di}erent distribution\ mainly in very small granules[ Bar is 4 mm[

while VAChT!LI was present in very small\ dust!like grains "Fig[ 2F#[ Accumulation of VAChT!LI in crush!operated sciatic nerves In crush!operated nerves the accumulation of VAChT! LI was already evident 0 h after crushing "Fig[ 0F#\ while the 9 h crushed nerves did not show any VAChT!LI "Fig[ 0E#[ The accumulations increased with time after the

operation\ as measured by the CFS!technique "Figs 4\ 5#[ The axons containing the VAChT!positive accumulated material were mainly large axons with intense immu! no~uorescence appearing not immediately proximal to the crush\ but at some distance[ These accumulated axonal segments were also synaptophysin!immu! noreactive "Fig[ 0#\ as well as SV1!positive "not shown#[ In addition\ more di}usely outlined groups of axons\ that represent autonomic unmyelinated axons could be barely observed to display any VAChT!LI[ These groups of

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352

Fig[ 3[ Confocal laser scanning photographs from rat tissue sections doubly incubated with anti!TH "left lane# and anti!VAChT "right lane#[ "A# Section from 7 h crush!operated "arrows# sciatic nerve of a normal rat[ Strongly ~uorescent TH!LI accumulations are present in axons close to the crush area[ Very few VAChT!containing axons "right lane# are found in this region\ but further proximal and distal to the crush\ VAChT!LI can be seen accumulated[ There is no colocalization between TH\ in sympathetic axons\ and VAChT\ mainly in motor axons[ Bar is 099 mm[ "B# Section from a 7 h crush!operated sciatic nerve of a rat which was lumbar sympathectomized 2 days before the crush operation[ No TH!positive axons can be seen[ However\ the number of VAChT!containing axons appear unchanged\ as compared with "A#[ Bar is 099 mm[ "C and D# Section of lumbar sympathetic ganglion\ showing a large number of TH!positive nerve cells\ surrounded by strongly VAChT!immunoreactive nerve terminals[ No clear VAChT!LI can be observed in any of the perikarya in the picture[ Bars are 099 mm and 49 mm\ respectively[

axons were\ however\ strongly synaptophysin immu! noreactive "Fig[ 0G# displayed accumulations just proxi! mal to the crush[ In sections double incubated with anti! VIP\ a weak VIP!immuno~uorescence was present in these groups of very weakly VAChT!positive _bres\ but occasionally also strongly immunoreactive VIP!positive axons were observed "Fig[ 2A#[ CFS!scannings revealed that the accumulation of VAChT!LI "in motor axons# proceeded with time\ and also retrograde accumulations were evident at 0Ð7 h "Fig[ 0G[ The retrograde accumulations were smaller than the proximal accumulations\ and measurements of this retro! grade fraction revealed that the ratio was about 39) of the anterograde accumulations "Fig[ 5#\ indicating a recycling of the VAChT\ probably in membraneous structures[ In comparison\ synaptophysin was recycling by about 69) "Fig[ 5#\ in agreement with previous inves! tigations "Li et al[\ 0881\ 0885a\ b#[ Accumulations of VIP!LI also increased with time after crushing\ but since

the strongly VIP!positive _bres in the rat sciatic were so few CFS scanning was not performed[

Discussion The vesicular acetylcholine transporter "VAChT#\ responsible for the accumulation of ACh inside synaptic storage vesicles\ has recently been functionally char! acterized "Erickson et al[\ 0883#[ The cDNA encoding for this protein has been cloned from several species includ! ing marine ray "Varoqui et al[\ 0883# rat and man "Erick! son et al[\ 0883#[ The enzyme that synthesizes ACh\ ChAT\ has been used as a marker for cholinergic neurons\ but since this enzyme also appears in non!neuronal tissue "Klapproth et al[\ 0866#\ it was not a good marker for cholinergic structures[ The gene that encodes for this enzyme has been located to the same chromosome that encodes for the VAChT\ and a {{cholinergic locus|| has

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Fig[ 4[ Rates of accumulation of VAChT!LI "A# and synaptophysin!LI "B# in rat sciatic nerve 0Ð7 h after crushing[ Cyto~uorimetric scanning curves[ Each point is the mean2SEM of 3Ð4 nerves "4Ð6 sections measured from each nerve#[ The two curves for proximal accumulations " _lled diamonds# are not exactly similar\ due to the fact that syn! aptophysin is present in other axons than the motor cholinergic VAChT!immunoreactive axons[ Distal to the crush\ the retrograde accumulations di}er somewhat in relative amounts accumulating distal to the crush^ more synaptophysin!LI appears to recycle than VAChT[

been identi_ed as a unique mammalian genomic arrange! ment "Gilmore et al[\ 0885#\ localized in the human to chromosome 09 "Strauss et al[\ 0880^ Roghani et al[\ 0883#[ The messenger RNA encoding for these two chol! inergic markers has been found by in situ hybridization to be co!expressed in the CNS and PNS at locations known to be cholinergic by pharmacological\ anatomical

and histological research[ In addition\ some sites have been identi_ed where mRNA for these two markers are present\ but which are not so far known to be cholinergic "Schafer et al[\ 0883#[ The VAChT protein\ a member of a large family of transmitter transporters\ has 01 transmembrane domains with both N! and C!terminal tails facing the cytoplasm\ but with glycosylated domains inside the vesicle[ It is the only transporter so far found that transports ACh\ after synthesis in the cytoplasm\ across the membrane to the inside of the storage:release vesicles[ Since this protein clearly identi_es cholinergic vesicles\ we used recently produced antibodies against VAChT to study the axonal transport of cholinergic vesicles in rat nerves[ In the motor neurons of the lower limb and sciatic nerve\ we found that the perikarya in the lumbar intu! mescence contained very little or undetectable amounts of the transporter\ while the nerve terminals in the muscle were strongly immunoreactive for VAChT[ This is similar to the pattern of other vesicle proteins studied^ Syn! aptophysin\ SV1\ synaptobrevin I and synaptotagmin II^ The cell bodies contained very low amounts\ probably due to rapid export of the formed vesicular elements into the axon "Li et al[\ 0885a\ b\ 0886#[ The motor endplates\ on the other hand\ were strongly immunoreactive for all the above vesicle proteins "Li et al[\ 0882\ 0885a\ b#\ and\ furthermore\ completely colocalized\ in high resolution confocal microscopy\ with VAChT!LI[ The CFS curves indicated that VAChT and the other vesicle peptides were moving with fast axonal transport anterogradely[ Distal to the crush\ where recycling material accumulated\ we found that VAChT was recycling by about 39)\ while synaptophysin\ as also described earlier\ recycled by about 69)[ It is not known which organelle the recycling material is stored in^ transmission EM has demonstrated multivesicular bodies and lamellated membranous struc! tures "Tsukita and Ishikawa\ 0879#\ but so far no immuno!EM has been carried out to identify these recyc! ling organelles[ However\ since the proportions of recyc! ling vesicle proteins is di}ering between e[g[ VAChT and synaptophysin\ this indicates that membrane modi! _cations take place in the nerve ending prior to recycling to the cell body\ perhaps at the endosome level "Sudhof\ 0884^ De Camilli and Takei\ 0885#[ In the autonomic cholinergic neurons in the lumbar sympathetic ganglia small amounts of VAChT!LI were present in a few perikarya\ which were\ in doubly incu! bated sections\ TH!negative[ A large number of strongly ~uorescent nerve terminals were seen basketing around virtually all perikarya\ including the large number of TH! positive cell bodies\ probably representing preganglionic nerve terminals[ The cell bodies with some VAChT!LI also contained VIP!LI[ In the nerve terminals in the hind! paw\ where the sympathetic cholinergic nerve terminals are projecting "Lundberg et al[\ 0868# we could see a large number of synaptophysin!immunoreactive nerve ter!

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Fig[ 5[ Cyto~uorimetric scanning curves from a sciatic nerve section\ 7 h after crushing\ doubly incubated with anti!VAChT "top _gure# and anti! synaptophysin "lower _gure#[ The corresponding ~uorescent micrographs are shown under the scanning graphs[ The number of axons with VAChT! immunoreactive material is clearly less than the number of synaptophysin!containing axons[ The distal accumulations were in this section for VAChT! LI 28[03) of the proximal accumulation\ and for synaptophysin 55[67) of the proximal accumulations[

minals\ but colocalized VAChT!LI was detected only in a few of these nerve terminals[ The VAChT!positive terminals were\ however\ also immunopositive for VIP as would be expected "Lundberg et al[\ 0868#[ In the crush operated sciatic nerve the unmyelinated autonomic axons contained surprisingly little accumu! lated VAChT!LI in contrast to the motor axons which displayed strong accumulations at some distance from the crush[ TH!positive axons were numerous in this region above the crush in normal rats\ but had disappeared almost totally after sympathectomy[ Some axons with rather weakly ~uorescent ChAT!immunoreactivity were observed in intact parts of the sciatic nerve\ indicating that also a fraction of this enzyme\ like TH\ is moving with fast transport\ probably by sticking to transported organelles "Dahlstrom and Li\ 0883#[ VIP!accumulations were generally rather weakly ~uorescent\ but occasional axons with strong immuno~uorescense could be seen[ In the few autonomic axons where a co!localization of VIP and VAChT could be seen\ high resolution confocal microscopy showed that the two antigens were dis! tributed in di}erently looking organelles^ VIP in large granules and VAChT present in small dust!like particles[

It was clear that the rat sciatic nerves contain much fewer cholinergic sympathetic axons than found earlier in the cat sciatic in experiments using anti!VIP and his! tochemical staining for AChE "Lundberg et al[\ 0868#[ The few cholinergic sympathetic axons found in this study on rat\ based on their content of VAChT!LI\ appear to have very varying amounts of the peptide VIP[

Conclusion We have shown that the vesicular ACh transporter VAChT is present in motor and sympathetic cholinergic neurons in the rat^ That this vesicle membrane protein is present in very low amounts in the perikarya "probably due to fast export into the axon#\ is transported with fast anterograde axonal transport in the axons together with other vesicle markers "e[g[ synaptophysin#\ and that the nerve endings contain high amounts of this protein\ as expected for a transporter in synaptic vesicles[ Some of the transporter protein is apparently recycled to the cell body\ since about 39) of the material found in ante!

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J[!Y[ Li et al[:Neurochem[ Int[ 21 "0887# 346Ð356

rograde accumulations were found in the retrograde accumulations distal to the crush[ Other vesicle proteins\ for instance\ synaptophysin had a recycling ratio much higher\ about 69)[ This may point to a membrane pro! cessing in the nerve terminal area prior to loading onto the retrograde transport machinery[

Acknowledgements Supported by the Swedish MRC "1196#\ EU grant no 85 0475^ the Royal Academy of Science and Arts in Got! eborg\ Gustav V]s 79!arsfond\ and by the Swedish Society for Medical Research[ For a generous supply of antibody preparations we are grateful to Dr R[ Jahn\ New Haven "p27#\ Dr R[ Kelly\ San Francisco "SV1#\ and Dr M[ Goldstein\ New York "anti!TH#[ The technical assistance of Ms Kerstin Lundmark is gratefully acknowledged[

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