SYNAPTIC VESICLE POOLS

Transcription

1 SYNAPTIC VESICLE POOLS Silvio O. Rizzoli* nd Willim J. Betz Astrct Communiction etween cells reches its highest degree of speciliztion t chemicl synpses. Some synpses tlk in whisper ; others shout. The louder the synpse, the more synptic vesicles re needed to mintin effective trnsmission, rnging from few hundred (whisperers) to nerly million (shouters). These vesicles reside in different s, which hve een given ewildering rry of nmes. In this review, we focus on five tissue preprtions in which synptic vesicle s hve een identified nd thoroughly chrcterized. We rgue tht, in ech preprtion, ech vesicle cn e ssigned to one of three distinct s. CALYX OF HELD SYNAPSE A gint nerve terminl in the uditory rinstem, it hs pivotl role in the circuitry tht is responsile for locting highfrequency sounds. *Mx-Plnck-Institut für iophysiklische Chemie, Am Fßerg 11, D Göttingen, Germny. Deprtment of Physiology nd Biophysics C-240, University of Colordo Medicl School, 4200 Est Ninth Avenue, Denver, Colordo 80262, USA. Correspondence to W.J.B. e-mil: ill.etz@uchsc.edu doi: /nrn1583 The defining feture of chemicl synpse is the collection of synptic vesicles in the presynptic terminl. These vesicles prticipte in cycle (for review, see REF. 1) tht permits them to e used repetedly during sustined ctivity. In typicl synpse t rest, smll percentge of vesicles re ttched to the presynptic memrne nd the rest reside in n djoining cluster. The synptic vesicles ll look like under the electron microscope, nd no significnt iochemicl distinctions re recognized tht might identify different clsses of vesicles in resting terminl. So, prt from the reltively few vesicles tht re ttched ( docked ) to the surfce memrne, the vst mjority seem to constitute single, homogeneous popultion. However, for decdes investigtors hve proposed tht there re distinct s popultions of vesicles whose memers possess distinct functionl properties. There is rther ewildering list of nmes for these s, including redily relesle, reserve, exo endo recycling, immeditely relesle, reluctnt nd resting. In this review, we will focus on the five preprtions tht hve een chrcterized most thoroughly with respect to vesicle s Drosophil lrvl neuromusculr junction (NMJ), frog NMJ, neontl rodent cultured hippocmpl neurons, neontl rodent CALYX OF HELD neurons in slice preprtions nd cutely isolted goldfish retinl ipolr cells. These preprtions spn rod phylogenetic rnge, permit diverse rry of reserch strtegies nd techniques, nd vry gretly in their ility to secrete qunt. Despite differences etween these preprtions, we propose tht it is possile to ssign every vesicle to one of three s, which we will cll the redily relesle (), the recycling nd the reserve. Erly oservtions of vesicle s The study of vesicle s egn with the work of Birks nd McIntosh 2,who investigted cetylcholine relese from ct sympthetic gngli (see lso REF. 3). They proposed tht there re two distinct presynptic stores of trnsmitter redily relesle frction, which is rpidly depleted t high frequencies of stimultion, nd non-redily relesle frction. Lter, Elmqvist, Qustel nd collegues 4,5 otined similr results from humn intercostl muscle studies. They found tht on high-frequency stimultion the mplitude of the responses decyed rpidly. They suggested tht the erly stimuli drew qunt from store of neurotrnsmitter from which qunt could e esily moilized the moiliztion store. The decy of the postsynptic response ws thought to e cused y this smll store not eing replenished fst enough during intense stimultion. Elmqvist nd Qustel greed tht the store of neurotrnsmitter corresponded to the redily relesle frction proposed y Birks nd McIntosh, nd tht the rest of the qunt represented the non-redily relesle frction. Most synpses rely on three vesicle s Elmqvist nd Qustel cutiously noted tht lthough the experimentl results re fully comptile with the [s] model, they cnnot e sid in ny wy to prove it 4.Since then, other possile explntions, including chnges in intrcellulr clcium concentrtion, prtil NATURE REVIEWS NEUROSCIENCE VOLUME 6 JANUARY

2 ACTIVE ZONE A portion of the presynptic memrne tht fces the postsynptic density cross the synptic cleft. It constitutes the site of synptic vesicle clustering, docking nd trnsmitter relese. FLASH PHOTOLYSIS Cells re dilysed with cged clcium compound (such s nitrophenyl-egta); ultrviolet flshes re then used to rek the cge to relese clcium. In this wy, the technique stimultes exocytosis independently of clcium entry from the extrcellulr spce. Recycling Time (s) Figure 1 Three vesicle s. The clssic three- model. The reserve mkes up ~80 90% of the totl, nd the recycling is significntly smller (~10 15%). The redily relesle () consists of few vesicles (~1%) tht seem to e docked nd primed for relese. Three kinetic components of relese (indicting relese of three vesicle s) on depolriztion of goldfish ipolr cells. The cell ws stimulted in the presence of the styryl dye FM 1-43, nd the increse in fluorescence gives direct mesure of exocytosis. Pnel modified, with permission, from REF. 12 (1999) Blckwell Scientific Pulishing. Chnge in fluorescence (%) relese from fully filled vesicles, full relese from prtilly filled vesicles nd desensitiztion nd/or sturtion of postsynptic receptors hve een investigted, ut were not found to fully explin the oserved chnges in relese. Therefore, through process of exclusion, the ide of distinct vesicle s tht possess different cpcities for exocytosis hs een strengthened. Although the terminology vries, most models (for exmple, FIG. 1) gree tht presynptic nerve terminls contin n, from which vesicles cn e esily moilized on stimultion, nd lrge reserve, from which vesicles re drwn more slowly, typiclly in response to intense or prolonged stimultion. A further distinction cn e mde, s not ll non- vesicles re eqully cple of eing relesed. Therefore, three s of vesicles hve to e postulted 6.The second of vesicles is relesed more slowly thn the, nd its relese precedes reserve moiliztion; for the purpose of this review we term this the recycling. One striking exmple of the relese of three vesicle s is shown in FIG. 1. Here, three different kinetic phses cn e oserved: rpid, lmost instntneous one, more prolonged slower one nd long-lsting phse. The properties of the three types re summrized in TABLE 1. The redily relesle. For the purposes of this review, we will define the s the synptic vesicles tht re immeditely ville on stimultion. These vesicles re generlly thought to e docked to the presynptic ACTIVE ZONE nd primed for relese, lthough it should e stressed tht docked vesicles re not ll necessrily immeditely relesle (see elow nd REF. 7). The is depleted rpidly y 5 15 shocks of highfrequency electricl stimultion 4,8 10, few milliseconds of depolriztion 11,12 or out 1 s of hypertonic shock in hippocmpl outons 13 (note tht the hypertonic shock is not physiologicl stimulus). Different sets of stimuli, such s high-frequency stimultion nd hypertonic shock 13, high-frequency stimultion nd clcium FLASH PHOTOLYSIS 10,or high frequency stimultion nd depolriztion 14,hve een shown to relese the sme vesicles. A further distinction cn e mde within the when relese is investigted in detil, in tht the vesicles might not ll e identicl in relese chrcteristics. At the clyx of Held it hs een shown tht the hs fst nd slow components of relese 15 (see elow). Also, in hippocmpl slices, Hnse nd Gustfsson suggest tht not ll vesicles tht re relesed during stimulus s short s 10 ction potentils (t 50 Hz) re identicl in relese prmeters. On verge, they found tht out two vesicles were relesed per ctive zone during stimultion. However, on verge only one of the vesicles ws found to e immeditely ville for relese 16. The recycling. We define the recycling s the of vesicles tht mintin relese on moderte (physiologicl) stimultion. This is thought to contin out 5 20% of ll vesicles. Physiologicl frequencies of stimultion cuse it to recycle continuously 8,19 21, nd it is refilled y newly recycled vesicles. One exception to this definition seems to occur in the goldfish ipolr nerve terminl, where it is difficult to scertin whether there re stimultion conditions tht would permit continuous recycling without reserve moiliztion. However, it should e stressed tht most reserch on this synpse hs een crried out using strong stimultion, such s continuous depolriztion 22 or clcium uncging 23. The reserve. The reserve is defined s depot of synptic vesicles from which relese is only triggered during intense stimultion. These vesicles constitute the mjority (typiclly ~80 90%) of vesicles in most presynptic terminls. The relese of these vesicles requires stimultion frequencies of t lest 5 10 Hz in frog NMJ 9,24,25, 30 Hz in Drosophil lrvl NMJ 26 or prolonged high potssium ppliction t the clyx of Held 21 nd possily lso t hippocmpl outons 27.It is possile tht these vesicles re seldom or never recruited during physiologicl ctivity. It is not cler wht triggers reserve relese, ut n experiment y Kuromi nd Kidokoro 28 indictes possile mechnism. They used the Drosophil temperture-sensitive mutnt shiire,which cnnot reform its vesicles t 34 C, ut functions normlly t room temperture. Stimultion t room temperture cused cycling of only the recycling. However, when recycling of these vesicles ws prevented t high temperture, vesicle relese continued from the reserve. Consistent with these results, t the frog NMJ lmost no reserve vesicles re relesed until the recycling is depleted 8,24.Therefore, it is tempting to suggest tht depletion of recycling vesicles triggers reserve moiliztion nd relese, lthough the underlying moleculr mechnisms remin oscure. 58 JANUARY 2005 VOLUME 6

3 Tle 1 Chrcteristics of the vesicle s Pool Redily relesle Recycling () Size (% of ~1 2% ~10 20% ~80 90% ll vesicles) Loction Docked Scttered Scttered (ulk of vesicle cluster) Relesed within <1 second A few seconds Tens of seconds, minutes Recycling Fst (seconds) Fst (seconds) (minutes) Mixing with Fst mixing with mixing mixing with other s recycling vesicles with reserve other vesicles Moility in resting None docked High Low (high in terminls ipolr cells) Vesicle s in five synptic systems Vesicle s hve een investigted in mny systems, using techniques such s electrophysiology nd electron nd fluorescence microscopy (BOX 1). The min chrcteristics of the vesicle s in five preprtions re summrized elow. Drosophil lrvl neuromusculr junction. The nerve terminl t the Drosophil NMJ (FIG. 2) contins ~84,000 qunt, 14 19% of which constitute the recycling 9. A rpidly depleted component of relese () ws oserved, with size of ~300 qunt 9. FM-dye stining indictes tht the recycling (sometimes referred to s the exo endo cycling 19,26,28 30 ) is typiclly found t the periphery of the outons. Hevy stimultion (30 Hz), ut not mild stimultion (10 Hz) or high potssium tretment, cn lso recruit vesicles from the reserve, which is found deeper within the outons 26.The recycling nd the reserve mix slowly, s the recycling cn e Box 1 Techniques used to determine vesicle chrcteristics Three sic techniques hve een used to mesure vesicle s electron microscopy, fluorescence microscopy nd electrophysiology. All three techniques hve een used, in some form, with ll five of the preprtions tht re discussed in this review: the frog neuromusculr junction (NMJ) 8,24,25,37,the Drosophil NMJ 9,26,28,29 31,139,neontl rodent cultured hippocmpl outons 13,20,38,40,130,the neontl rodent clyx of Held presynptic terminl 10,14,21,50,64,65,157 nd the goldfish retinl ipolr nerve terminl 12,71,72,74,79,80,129.Electron microscopy studies hve included freeze frcture microscopy nd trnsmission microscopy, sometimes involving seril reconstructions nd the use of endocytic mrkers to lel vesicles. Fluorescence microscopy hs relied primrily on styryl dyes (FM dyes), the presynptic uptke nd relese of which cn e monitored in living preprtions in reltime 73.In FM-dye experiments, the synptic preprtion is thed in solution contining the dye, nd stimulted to induce exocytosis. The vesicles open to the extrcellulr solution nd their internl memrne leflets re lelled with the dye. When endocytosis is complete, the newly endocytosed synptic vesicles remin lelled nd cn e imged (cell memrnes re wshed to remove the dye). Electrophysiologicl studies hve included recordings from postsynptic cells, nd, in the cse of the clyx of Held neurons nd goldfish ipolr cells, presynptic whole-cell voltge clmp, including ptch-clmp cpcitnce. Other preprtions hve een used to chrcterize vesicle s, including NMJs of the snke 161,162 nd cryfish 163, Aplysi clifornic neurons 164,the squid gint synpse 165 nd lmprey reticulospinl synpses 116. loded nd unloded y mild stimultion without reserve moiliztion 19,28.The recycling cycles rpidly during stimultion, wheres the reserve recycles more slowly fter the cesstion of 30 Hz stimultion 30. An unusul feture of Drosophil terminls concerns the trffic in the centre of the outons, which is populted y reserve vesicles. FM stining is oserved here fter recovery from totl vesicle loss in shiire mutnts 28,or fter high-frequency stimultion 26 nd certin drug tretments 29, nd FM dye relese from the centres hs een noted, indicting vesicle moiliztion nd exocytosis 19,26.However, vesicles evidently remin here only trnsiently, moving out to the periphery over time. So, in resting preprtions, outon centres re lmost devoid of smll vesicles 31 (FIG. 2;see REF. 32 for different result). Moreover, synptotgmin (nd presumly, therefore, synptic vesicle) immunofluorescence in this preprtion is typiclly confined to the periphery of the outon 33.Consistent with the ide of trfficking, outon centres were not devoid of vesicles during recovery from totl vesicle loss in shiire mutnts 34.Movement of FM fluorescence in the opposite direction, from the periphery of the outons to the centrl region, hs lso een oserved 30. In summry, it seems tht the centrl regions of Drosophil outons re populted y the reserve vesicles for only rief period fter hevy depletion-inducing stimultion, nd they re rpidly removed to the periphery of the outons. So, the distriution of the recycling nd reserve vesicles might depend on the conditions under which the preprtions were oserved (H. Kuromi, personl communiction). Ultrstructurl (FM-dye photoconversion) oservtions of Drosophil preprtions under vrious conditions would e useful. Frog neuromusculr junction. In the cutneous pectoris nd srtorius NMJs of the frog (Rn pipiens) (FIG. 3), the totl vesicle contins ~500,000 vesicles 25,35,36.An initil phse of relese (from the ) is exhusted within ~0.5 s of high-frequency stimultion 8 ; these vesicles mke up ~2% of the totl 37. The recycling cycles continuously t 2 Hz stimultion (tht is, the reserve is not moilized), ut it is depleted within ~10 s of high-frequency (30 Hz) stimultion. The recycling mkes up ~10 20% of the totl vesicle popultion 8,24,37.At the frog NMJ, the is evidently n integrl prt of the recycling, s the rte of recovery depends on the rte of refilling of the recycling 8.The reserve, which only strts to dischrge fter s of 30 Hz stimultion, recycles slowly, with time constnt of severl minutes (FIG. 3e, lue rrows). The mixing etween the recycling nd reserve s (red rrow) is slow, in the order of hours 24. Hippocmpl outons. In cultured hippocmpl neurons (FIG. 4), the totl vesicle popultion in ech outon comprises vesicles 38 (studies in hippocmpl slices hve yielded lrger vlues 39 ). An immeditely ville vesicle (), contining ~5 20 vesicles 40,is relesed y hypertonic shock 13,41, nd is proly the NATURE REVIEWS NEUROSCIENCE VOLUME 6 JANUARY

4 c v 1 m 10 µm 500 nm 4 µm d e 0 s 300 s ~70,000 ~80% ~14, % Fst ~300 ~0.4% 80 s 100 s 300 na 300 na Recycling Figure 2 Typicl imges from the Drosophil lrvl neuromusculr junction preprtion. FM 1-43 fluorescence imge of nerve terminl; note the fluorescent outons. Cross-section of outon; rrows indicte ctive zones; m, mitochondrion; v, synptic vesicles; 1, xon. c Three-dimensionl reconstruction of two Drosophil lrvl xons; the drk ptches represent synpses; the line seprtes the two muscles innervted y the xons. d Postsynptic current recording from muscle under continuous stimultion t 10 Hz (top); few individul postsynptic responses t 0 s nd 300 s fter stimultion commenced (ottom). The redily relesle () is depleted in the first few shocks relese cn then e mintined lmost indefinitely y repeted recycling of the recycling t this frequency. e Pool sizes nd mixing rtes. Blue rrows indicte endocytosis; red rrows indicte mixing etween s. Red sphere indictes totl size reltive to the other preprtions; na, nnoamp. Pnels nd c modified, with permission, from REF. 31 (1993) John Wiley & Sons, Inc. Pnel d modified, with permission, from REF. 9 (2000) Elsevier Science. sme of vesicles tht is relesed within ~2 s of 20 Hz stimultion 13,40,42.A of vesicles tht recycles repetedly with stimultion (recycling 20,43 45 ) comprises 10 20% of the vesicles 20 (for review, see REF. 43). The rest of the vesicles (reserve ) were reluctnt to undergo exocytosis in response to oth electricl stimultion nd (short) high potssium ppliction 20,43. Occsionlly, vesicles other thn those contined in the nd recycling could e lelled 45,46,ut it is not cler whether this is consistent phenotype. Why re reserve vesicles in hippocmpl outons so difficult to relese? One possiility is tht the stimultion protocols tht were used in hippocmpl studies permit the recycling to e refilled dequtely y newly retrieved vesicles, so the reserve vesicles remin in plce. Mixing etween the recycling nd the reserve s seems to e slow, wheres mixing etween the nd the recycling is reltively fst. The recycling (nd ) vesicles re rpidly retrieved y endocytosis fter stimultion, through mechnisms tht do not seem to require endosoml intermedites (for reviews, see REFS 43,45). The reserve vesicles, when relesed, proly recycle slowly 27.An ultrfst mode of endocytosis kiss-nd-run 47,48 (see elow) might lso tke plce t this synpse, t lest in the cse of the vesicles 44,49. Clyx of Held presynptic terminl. The totl numer of vesicles t this synpse in the rt (FIG. 5) ws estimted in recent electron microscopy study 50 to e t lest 70,000 qunt. A higher estimte ~188,000 vesicles ws otined in nother study 21. Relese from the clyx is depressed rpidly with stimultion y high-frequency pulses (5 300 Hz (REFS 51,52)). Furthermore, finite of synptic vesicles is rpidly relesed on depolriztion or clcium uncging 14,15, The consists of ~1,500 4,000 vesicles in the postntl dy 8 11 clyx of Held nerve terminl 15,54, Two distinct components of relese hve een oserved for the : fst component (~3 ms time constnt of relese 15 ) nd slow one (~30 ms), ech mking up out hlf of the 15,68.Interestingly, the fst component of relese recovers more slowly (within seconds) fter depletion thn the slow relese component (within 100 ms) 15. The underlying cuses of the existence of the two components hve not yet een resolved 69.It is possile tht the fst component of relese consists of those vesicles tht re situted closest to clcium chnnels, nd the slow component of vesicles situted further wy 70.It is tempting to propose tht certin stimuli, such s highfrequency trins of ction potentils, relese only one component of the (the fst component), s some mesurements of the size using high-frequency stimultion show reltively low vlues It should e noted tht mesuring vesicle prmeters in the clyx of Held through nlysis of postsynptic responses is y no mens trivil study 58,66,67.When the clyx nerve terminls were stimulted t 5 Hz for up to 20 min, or t 20 Hz for up to 5 min in the presence of n FM dye, ~5% of ll vesicles were lelled 21.This indictes tht the recycling consists of ~10,000 vesicles (lthough higher estimte of ~20,000 vesicles ws clculted using certin ssumptions on vesicle relese t this synpse 21 ). High potssium stimultion cused lrger percentge of vesicles to recycle (for exmple, 30% for 15 min of stimultion), drwing vesicles from the reserve 21. The mixing rte etween the nd the recycling is uncler. As the recovers rpidly (time constnt ~1 s (REFS 14,15)) it is likely tht it is refilled through moiliztion of vesicles from the recycling. Therefore, mixing of the two s would e fst, t lest on stimultion. The mixing etween the recycling nd the reserve seems to e slow, t lest with physiologicl stimultion, s the reserve vesicles re reluctnt to relese 21. endosoml intermedite-dependent endocytosis recycles reserve vesicles, ut fster route functions for vesicles from the recycling JANUARY 2005 VOLUME 6

5 c FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING (FRAP). A method used to mesure the lterl diffusion of elements. It requires tgging of the molecule of interest with fluorescent mrker, photoleching of the lel with pulse of lser light nd susequent mesure of the rte of fluorescence recovery into the leched re s other lelled molecules move into it. 10 µm 0.5 µm 0.5 µm d 0 s 50 s e ~400,000 ~80% ~75,000 ~10 20% Fst ~10,000 ~2% 10 s 2 mv 2 mv 50 ms Recycling Figure 3 Typicl imges from the frog neuromusculr junction preprtion. FM 1-43 fluorescence imge of nerve terminl. Electron microgrph of cross-section through the nerve terminl. Arrows indicte ctive zone. c Three-dimensionl reconstruction of n pproximtely 2 µm-long nerve terminl segment. Active zones re shown in red. d Postsynptic potentil recording under continuous stimultion t 30 Hz (top); few postsynptic responses t 0 s nd 50 s fter stimultion commenced re shown elow (imge courtesy of D. A. Richrds). In the ottom trce, verticl lines re shock rtefcts; synptic responses hve declined to seline. The is rpidly exhusted, followed y recycling depletion. relese is mintined for t lest one minute. e Pool sizes nd mixing rtes. Blue rrows indicte endocytosis; red rrows indicte mixing etween s. Red sphere indictes totl size reltive to the other preprtions. Pnel c reproduced, with permission, from REF. 37 (2004) Americn Assocition for the Advncement of Science. Goldfish retinl ipolr nerve terminls. Estimtes from electron microscopy plce the totl of synptic vesicles in the goldfish retinl ipolr nerve terminl (FIG. 6) t round 700,000 qunt (~500,000 or 900,000 qunt in smll nd lrge nerve terminl, respectively 71 ;see REF. 72 for lower estimte), consistent with styryl dye imging experiments 73,74. When exocytosis ws proed using styryl dye imging 12 (FIG. 1) or interference reflection microscopy 75, three s of vesicles were indicted y three kinetic phses of relese. A redily relesle component ws estimted (typiclly from rief (tens of milliseconds) depolriztion) t 1,000 1,800 vesicles 11,12,23,76,77.Longer depolriztion pulses (ut typiclly less thn 1 s) relesed second of ~3,000 4,400 vesicles 12, The two s dd up to ~6,000 vesicles in mny studies 79,80.The numer of vesicles in these two s, which together ccount for only 1 2% of the totl vesicle popultion, corresponds to the numer of vesicles tht re ttched to rions structures tht re thought to collect vesicles from the cytosol nd guide them to the relese sites 81 (see REF. 82 for different interprettion). Continuous depolriztion seems to continuously drw vesicles from lrge reserve 12,74. The mixing rtes etween the s hve not een investigted in detil, ut, fter exocytosis, the vesicles on the rions seem to e replenished rpidly y reserve vesicles, rther thn y recycling vesicles, which indictes rpid mixing 72.Both fst nd slow recycling mechnisms might return vesicles to the reserve (see elow). FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING (FRAP) experiments in goldfish ipolr nerve terminls 72 nd cone terminls of the nole lizrd retin 83 hve shown tht vesicles in the cytoplsm of resting terminls re highly moile, which might reflect lck of synpsin in these rion synpses(see elow). Summry of size. In the four non-rion synpses, the extensive rnge of vesicle sizes cn e unified y normlizing to the totl length of ctive zone mteril 84.The totl numer of synptic vesicles per ctive zone in the Drosophil NMJ is 87,000 vesicles, which, divided etween 400 ctive zones 31,is equl to 217 vesicles per ctive zone; in hippocmpl outon there re 200 vesicles per ctive zone 38 ; nd in the clyx of Held terminl there re 200,000 vesicles for 600 ctive zones 50,85,giving n verge of 333 per ctive zone. At the frog NMJ, the ctive zones re out six times longer thn in the other preprtions, perhps reflecting oligomeriztion of the smller units. On this sis, 500,000 vesicles shred etween ctive zones equls 270 vesicles per ctive zone. The goldfish ipolr cell, with 700,000 vesicles nd 60 rions, gives very different result: more thn 11,000 vesicles per rion 71. Similr results re otined when the numer of docked vesicles t ech ctive zone is considered: ~5 10 vesicles in hippocmpl outons 40, ~2 vesicles in the clyx of Held 50 nd ~40 vesicles per long ctive zone in the frog NMJ, which, eing out six times longer thn the other ctive zones, gives out 7 vesicles for the equivlent re (S.O.R. nd W.J.B., unpulished oservtions). In the goldfish ipolr terminl ech rion tethers ~110 vesicles 71,76. Vesicle s re not ntomiclly segregted The schemtic digrm in FIG. 1 is typicl of those tht hve een drwn for severl decdes, showing the three vesicle s morphologiclly segregted into distinct clusters. This digrm implies tht the further the cluster is from the presynptic memrne, the slower it is to relese. Until recently, there ws no direct evidence to either support or refute this model. The vesicles in the must, y definition, e le to undergo exocytosis immeditely fter stimultion, so must lie t or close to the presynptic memrne. However, the vesicles tht mke up the other s re recruited t more leisurely pce during repetitive stimultion, nd could, in principle, e trnsported to NATURE REVIEWS NEUROSCIENCE VOLUME 6 JANUARY

6 c 0.5 µm Axon Mitochondrion SNARE PROTEINS A fmily of memrne-tethered coiled-coil proteins tht re required for memrne fusion in exocytosis (such s during neurotrnsmitter relese) nd other memrne trnsport events. When trns-snare complexes re formed etween vesicle SNAREs nd trgetmemrne SNAREs, they pull the two memrnes together, presumly cusing them to fuse. Bouton 5 µm Mitochondrion Dendrite Dendrite 0.1 µm Sucrose Fst ~170 ~85% ~20 ~10% 5 pa relese sites from ny loction within the entire vesicle popultion. Recent evidence indictes tht, t lest in some preprtions, vesicles in the recycling nd reserve s re intermixed to considerle degree. As the cn e depleted within few milliseconds or tens of milliseconds with strong stimuli such s clcium uncging 23 or depolriztion 15,59,60,65,vesicles elonging to the must e docked t or lie very close to the ctive zones 86,87 nd consequently to voltgegted clcium chnnels 88 nd they would lso hve their SNARE PROTEINS in relese-redy configurtion 89. The ctive zone mchinery intercts in complex mnner with the morphologiclly docked vesicles 90,which might promote interction of vesiculr components with clcium chnnels 91,resulting in rpid relese on stimultion. 1 s Fst ~10 ~5% Recycling Figure 4 Typicl imges from the rt cultured hippocmpl preprtion. FM 1-43 fluorescence imge of field showing numerous lelled presynptic outons. Electron microgrph of cross-section through outon. Arrowheds indicte the two edges of the ctive zone in this imge; the lck rrows point to two docked vesicles; non-docked vesicle ner the ctive zone is shown y the white rrow. c Three-dimensionl reconstruction of outon. d Postsynptic response to hypertonic sucrose ppliction (r); this tretment selectively releses the. pa, picoamp. e Pool sizes nd mixing rtes. Blue rrows indicte endocytosis; red rrows indicte mixing etween s. Red sphere indictes totl size reltive to the other preprtions. Pnel modified, with permission, from REF. 166 (2001) Elsevier Science. Pnel c reproduced, with permission, from REF. 40 (2001) Mcmilln Mgzines Ltd. Pnel d modified, with permission, from REF. 13 (1996) Elsevier Science. d e Consistent with this interprettion, the numer of docked vesicles in some preprtions is similr to the numer of redily relesle vesicles 50,71.In hippocmpl outons, when vesicles relesed y short tetnic stimultion were lelled with n FM dye nd counted under the electron microscope, the numer of lelled vesicles correlted closely with the totl numer of docked vesicles in the sme terminl (lthough there is evidence tht not ll docked vesicles re esily relesle, so some might not elong to the 37,92 ). The fct tht recycling vesicles recycle selectively without reserve relese ment tht they could e lelled selectively, nd their position ws investigted using electron microscopy. In hippocmpl outons 20,43, the clyx of Held 21 nd the frog NMJ 37 (FIG. 7),the recycling ws scttered throughout the nerve terminls under stimultion conditions tht triggered continuous recycling. In Drosophil NMJs, fluorescence microscopy showed tht the recycling occupies the periphery of the synptic outons 28, nd similrly, t the frog NMJ, recycling vesicles tended to e excluded from the cores of the vesicle clusters 37.The reserve ccounts for most of the vesicle cluster in ech preprtion. At the frog NMJ, the reserve vesicles did not seem to e prticulrly excluded from ner-ctive zone sites 37, lthough reltively more vesicles were found in this position. In lmprey reticulospinl synpses reserve vesicles seem to form the ulk of the vesicle clusters tht re distnt from the ctive zone 93. How re vesicles clustered? In resting terminls most synptic vesicles re immoile 94,95, 131.Synpsin is the oldest, nd still the est, cndidte for the glue tht inds them together 96, lthough severl studies hve prompted importnt djustments to the originl synpsin hypothesis, which ws sed minly on oservtions of synpsin locliztion on the cytoplsmic surfce of synptic vesicle memrnes 96, nd the reduced ffinity for vesicle inding fter phosphoryltion of synpsin y clcium/clmodulin-dependent protein kinse II (CMKII (REFS 97,98)). Vesicles in the lmprey reticulospinl synpse tht were distnt from the presynptic memrne were specificlly lost fter cute nti-synpsin ntiody injection 93, nd similr phenomenon ws oserved in mice lcking synpsin I (REFS 99,100).Also lost with the synpsin-positive ws the ility to sustin high-frequency relese, lthough low-frequency relese ws mintined 93. These oservtions led to the suggestion tht synpsin holds vesicles together specificlly in the reserve 96. However, other oservtions show tht synpsin molecules do not lwys discriminte etween reserve nd non-reserve vesicles. For exmple, synpsin dispersion from vesicle clusters hs een oserved with stimultion tht triggers recycling -only relese 101. Moreover, synpsin knockouts in mice not only reduce the overll numer of vesicles 99,102,ut cn lso ffect the size of the recycling 103 or the 104.Furthermore, rpid depression of relese (proly not relted to reserve moiliztion) hs een oserved in synpsin I/II knockouts 102.In ddition, the complexity 62 JANUARY 2005 VOLUME 6

7 c 10 µm 5 µm 0.3 µm 10 µm d e f Cm Fst 2 mm Clcium 4 mm Clcium 10 ms ~180,000 95% ~7,000 ~4% ~3,000 ~1% 3 na 10 & 30 ms 5 ms 2 ms 200 ff 1 ms 10 ms Recycling Figure 5 Typicl imges from the rt clyx of Held preprtion. FM 1-43 fluorescence imge of single identified clyx of Held nerve terminl. Top: low power cross-section of the preprtion; the presynptic terminl is yellow; the postsynptic cell is lue; nd the postsynptic nucleus is red. Bottom: higher power imge (see ox in top pnel); rrows indicte ctive zones. c Three dimensionl reconstruction (presynptic terminl in ornge; postsynptic cell in lue). d Postsynptic current recording under 100 Hz stimultion; the is rpidly depleted. e Presynptic cpcitnce response to short (1 30 ms) depolriztion; the response (indictive of redily relesle exocytosis) plteus t ~10 ms of depolriztion. The gp etween seline nd response to stimultion cn e ttriuted to the fct tht cpcitnce recording is not relile during stimultion. Cm, memrne cpcitnce; ff, femtofrd. f Pool sizes nd mixing rtes. Blue rrows indicte endocytosis; red rrows indicte mixing etween s. Red sphere indictes totl size reltive to the other preprtions. Pnel reproduced, with permission, from REF. 21 (2003) Society for Neuroscience. Pnels nd c reproduced, with permission, from REF. 50 (2002) Society for Neuroscience. Pnel d modified, with permission, from REF. 10 (1999) Elsevier Science. Pnel e modified, with permission, from REF. 65 (2001) Elsevier Science. of the possile roles of phosphoryltion is underscored y the identifiction of different kinses tht phosphorylte synpsin in vivo 105, nd the report tht CMKII knockouts show incresed rther thn decresed trnsmitter relese 106. All three synpsin genes hve een knocked out, singly nd in comintion 99,100,103,107,108.None of the muttions, including the triple knockout (P. Greengrd, personl communiction), re lethl. Similrly, Drosophil mutnts tht lck ll synpsins re vile 109. These results re not fully explined y ny simple model. For exmple, in synpsin I knockouts, synptic depression is incresed 102 nd FM-dye uptke is decresed 103,wheres the opposite hs een reported for synpsin III knockouts 110. In two rion synpses, including the goldfish ipolr nerve terminl, reserve vesicles re highly moile 72,83 nd the first vesicles to e relesed re immoilized on rions, in contrst to the sitution in conventionl synpses. Interestingly, rion synpses lck synpsin, nd the uninhiited movement of reserve vesicles is dequte to replce vesicles on the rions 72.However, it should e noted tht ultrstructurl oservtions hve shown structures tht interlink vesicles in rion synpses 111. If recycling vesicles in non-rion synpses re defined y their reltive lck of synpsin crosslinks, some sort of cge must prevent their dispersl nd consequent loss from the cluster. One cndidte for this role is ctin, which tightly surrounds vesicle clusters in some synpses 112,113.However,perturtion of ctin dynmics does not noticely ffect the overll FM stining pttern of the vesicle clusters It is lso possile to visulize model in which ll vesicles re eqully moile, ut only some (recycling ) cn exocytose efficiently on interction with the ctive zone. This model would explin the lck of ntomicl segregtion etween the vesicle s, nd it fits well with the dt from mmmlin synpses, where the reserve vesicles re reluctnt to relese, nd might, therefore, hve limited fusion competence. However, it does not explin systems such s the frog nd Drosophil NMJs, in which the reserve vesicles re perfectly relese-competent ut re moilized only fter recycling depletion. How re vesicles in different s moilized? As discussed ove, vesicles re thought to e docked t the ctive zones, so would not need to move to e relesed. By contrst, the recycling vesicles re not necessrily found in the vicinity of the ctive zones. At lest two models might explin their exocytosis: directed movement towrds the ctive zones nd rndom diffusion. In the first model, the recycling vesicles would require ccess to cytoskeletl trcks tht guide them to the ctive zone; in the second, simple diffusion could e sufficient to llow these vesicles to rech the ctive zone (FIG. 7d). Wht cytoskeletl elements could guide vesicles towrds the relese sites? The ctin cytoskeleton is undnt t motor nerve terminls 112,115,where it minly surrounds synptic vesicle clusters 112,113,116,117. Notly less ctin is found within the clusters, lthough filments resemling ctin hve een oserved in ultrstructurl studies 118.Actin filments tke prt in orgnelle movement in vrious systems 119,120, nd there is strong evidence for the involvement of ctin in the trnsport of synptic vesicles ck to vesicle clusters fter endocytosis in lmprey reticulospinl synpses 116,121 nd cultured cells 122 ut not in hippocmpl outons 113. NATURE REVIEWS NEUROSCIENCE VOLUME 6 JANUARY

8 10 µm 3 µm 300 nm c d e 0.5 ms pulse Fst ~700,000 ~99% ~4,400 ~0.6% 25 ms pulse ~1,200 ~0.17% 2 µm 10fF 0.5 s Figure 6 Typicl imges from the dissocited goldfish retinl ipolr cell preprtion. FM 1-43 fluorescence imge; the dye decortes the whole cell (see rightfield imge in inset), ut is only internlized in the nerve terminl. Cross section of the nerve terminl; top, low power imge. c Three-dimensionl reconstruction of nerve terminl; ctive zones re indicted y red circles. d Presynptic cpcitnce responses to short depolriztion (left, 0.5 ms, nd right, 25 ms). Only vesicles re relesed y such rief stimuli; the response plteus t ~20 ms of depolriztion, indicting totl depletion of the. The decy of the signl indictes endocytosis. e Pool sizes nd mixing rtes. Blue rrows indicte endocytosis. Red sphere indictes totl size reltive to the other preprtions. Pnel reproduced, with permission, from REF. 74 (1996) Elsevier Science. Pnel reproduced, with permission, from REF. 72 (2004) Elsevier Science. Pnel c reproduced, with permission, from REF. 71 (1996) Elsevier Science. Pnel d modified, with permission, from REF. 11 (1996) Elsevier Science. Actin disruption experiments produced wide spectrum of results, including depression of relese t Drosophil lrvl, frog nd snke NMJs 28,112,115, t the clyx of Held 123 nd in chromffin cells 124 ; no chnge in relese prmeters t goldfish ipolr terminls 114,125 ; nd slight potentition of relese t hippocmpl outons 113,126.The effects of ctin disruption might lso depend on the ge of the synpse, with vesicle cycling ffected less in mture synpses 127.Blocking ctomyosin movement with gents tht inhiit myosin light-chin kinse hmpers relese in hippocmpl outons 128.It is importnt to note tht reserve relese, ut not recycling relese, seems to e impeded y ctin disruption t the Drosophil NMJ 26. Recycling of synptic vesicle s Selective recycling to the of origin. Do vesicle s tend to mintin their tgs throughout synptic vesicle recycling, or is there mixing etween s? To explore this question we will consider the three s in turn. At the frog NMJ, recovery of the depends on refilling of the recycling, indicting tht the vesicles represent supopultion of the recycling 8, nd tht recycling vesicles proly lose their sttus. At goldfish ipolr nerve terminls, the newly recycled vesicles refill the reserve 72,129,nd the vesicles on the rions do not recycle selectively. Recycling of the hs not een investigted in Drosophil. In hippocmpl outons, series of elegnt experiments comining electrophysiology nd styryl dye imging showed tht vesicles cn e relesed repetedly 130 (FIG. 8).Pyle nd collegues estimted tht the mixes with the recycling in out 20 s if the is depleted electriclly, or in out 5 min if hypertonic shock is used. The fte of recycling vesicles t the frog NMJ hs een investigted using severl experimentl models, ll of which indicte tht recycling vesicles selectively return to their of origin. In one experiment (FIG. 8), fully loded terminl ws uzzed (riefly stimulted t high rte) to relese the recycling. The vesicles lost their dye nd recycled. After 15 min, the preprtion ws destined using prolonged highfrequency stimultion. When the stimultion egn, little or no dye ws lost (even though the terminl ws secreting norml numer of qunt); the uzz-recycled, dye-empty vesicles hd returned selectively to the recycling. This result lso shows tht the recycling nd reserve s operte sequentilly: reserve vesicles re not relesed in lrge numers until the recycling is depleted. Recycling vesicles cycle repetedly, without reserve relese, during prolonged physiologicl stimultion t the frog NMJ 8, Drosophil NMJ (for review, see REF. 19), hippocmpl outons 20,43 nd the clyx of Held 21.This consistent oservtion is proly the strongest piece of evidence for the existence of distinct vesicle s. Recycling routes for different s. Two clssic studies of synptic vesicle recycling, involving electrophysiology nd electron microscopy, were undertken t the frog NMJ in the erly 1970s (REFS 25,35,131,132).Heuser nd Reese 25 showed tht high-frequency stimultion resulted in vesicle depletion, nd tht vesicle reformtion required endocytic intermedites. Using low-frequency stimultion, Ceccrelli nd collortors 35 found no significnt vesicle depletion, nd no requirement for endocytic intermedites. More recent experiments on this preprtion 8,24 hve shown tht stimuli tht relese the reserve result in slow endocytosis tht proceeds through the formtion of infoldings, wheres when the recycling ws selectively relesed no such intermedites were oserved. The recycling vesicles 64 JANUARY 2005 VOLUME 6

9 500 nm c Control shiire 300 na 80 s Synptic current (normlized) Frction relese tht is recycled (per shock) Control Control minus shiire=recycled vesicles shiire Seconds t 10 Hz d Actin Recycling Figure 7 Topology, recycling nd moiliztion of vesicle s. The recycling is scttered throughout the vesicle cluster. Single cross section through (left) nd three-dimensionl reconstruction of (right) frog motor nerve terminls in which the recycling ws selectively lelled. Proposed recycling model. The reserve (pink) recycles slowly, through formtion of infoldings nd their rek-off through clthrin cot-dependent mechnisms. The recycling (purple) cycles through direct endocytosis from the plsm memrne. This process might e clthrin cot dependent, or might rely on trnsient fusion (kissnd-run). Kiss-nd-run might e used especilly y redily relesle () vesicles. c Newly recycled vesicles mintin most vesicle relese t Drosophil synpses. Left: postsynptic recording t non-permissive temperture in control neuromusculr junctions (NMJs) or shiire mutnt NMJs t 10 Hz of stimultion. The relese is mintined lmost indefinitely in controls, ut it decys rpidly in the mutnt. Right: comprison of the relese for the two conditions. The difference etween the two is shown in green. The lue line shows the frction of the relese (per shock) tht is performed y newly recycled vesicles. d Proposed model of moiliztion. The vesicles re docked nd do not require moiliztion. The reserve vesicles form most of the cluster nd re tightly crosslinked, possily y synpsin. The recycling vesicles re not s hevily crosslinked, so re more moile. They might e le to diffuse to the ctive zone (rrows, left). Alterntively (right), they might hve ccess to cytoskeletl elements (for exmple, ctin 118 ) tht direct them towrds the ctive zone. Pnel reproduced, with permission, from REF. 37 (2004) Americn Assocition for the Advncement of Science. Pnel c reproduced, with permission, from REF. 9 (2004) Elsevier Science. CLATHRIN A vitl structurl component of coted vesicles tht re implicted in protein trnsport. Clthrin hevy nd light chins form triskelion, the min uilding element of clthrin cots. DOMINANT NEGATIVE A mutnt molecule tht cn form heteromeric complex with the norml molecule, knocking out the ctivity of the entire complex. seem to endocytose directly from the plsm memrne, proly through CLATHRIN-cot-dependent mechnisms 37.So, two vesicle recycling routes cn e envisged ulk endocytosis, nd direct endocytosis from the plsm memrne (FIG.7) depending on the strength of stimultion. endocytosis (of reserve vesicles) might lso require endosoml sorting of vesicle components, s originlly proposed y Heuser nd Reese 25.The smll GTPse R5 is present minly on erly endosomes nd, through interction with severl effectors, regultes trffic through this comprtment 135.R5 ws found on synptic vesicles 134,135, nd overexpression of R5 resulted in the formtion of norml endosomes in the xons of cultured hippocmpl neurons 135.Moreover, when R5 function ws pertured in Drosophil motor nerve terminls y expression of DOMINANT-NEGATIVE mutnt form, vesicle cycling ws disrupted, with relese nd endocytosis eing inhiited, nd ccumultion of endosome-like structures in the terminls 136.In ddition, the endosoml SNARE Vti1 is enriched on synptic vesicles, where it is prt of SNARE complex (ut not the exocytic complex 137 ). Similr fst nd slow recycling pthwys hve een shown in ultrstructurl studies in the dult Drosophil NMJ nd hve lso een indicted y fluorescence studies in the Drosophil lrvl NMJ 30.In mmmlin synpses, reserve vesicles re reluctnt to relese, so it ws not surprising tht the originl styryl dye uptke studies t this synpse indicted single recycling pthwy. This pthwy recovered vesicles with hlf time of ~20 s, consistent with vesicles fusing with the plsm memrne nd eing retrieved through clthrin-medited mechnism. A more direct investigtion using FM-dye photoconversion showed limited ulk uptke 20,43,45.Also, the vesicles were found to mintin their identity throughout vesicle recycling 145,providing further evidence ginst ulk endocytosis. This is consistent with the strong resistnce of hippocmpl synpses to vesicle depletion (compre REFS 146 nd 147). When prolonged stimultion (mny minutes) y high potssium depolriztion ws used in mmmlin synpses 21,27,endocytic intermedites tht were indictive of ulk endosoml uptke were evident. So, it is possile tht when the normlly reluctnt reserve vesicles re relesed, they endocytose through slow pthwy. One NATURE REVIEWS NEUROSCIENCE VOLUME 6 JANUARY

10 Sucrose Sucrose Stimultion Dye 'Buzz' Destin EPSC FM pa Fluorescence (% initil) Control (no uzz) Buzz Seconds Figure 8 Redily relesle nd recycling vesicles cn recycle selectively. The recycling in hippocmpl outons ws loded with the styryl dye FM 2-10, nd the redily relesle () ws repetedly relesed y sucrose ppliction; this ws mesured y postsynptic recording (top) or y fluorescence relese (ottom). Neurotrnsmitter relese recovered rpidly etween sucrose pplictions, ut different result ws otined for fluorescence relese, indicting tht newly recycled, FM-empty vesicles were relesed on the second sucrose ppliction. Therefore, the recycles selectively, ut the mixing of the nd recycling is fst (seconds to minutes). EPSC, excittory postsynptic current; pa, picoamp. Both the recycling nd the reserve were lelled in frog NMJ terminls. The terminls were riefly stimulted ( uzzed ) to relese the recycling, nd the terminls were then rested for few minutes. The preprtions were then destined. Note tht dyeempty (recycling ) vesicles re relesed during the first ~10 s of stimultion (dotted line), indicting tht the recycling vesicles relesed during the uzz hve recycled selectively to their of origin. Pnel modified, with permission, from REF. 130 (2000) Elsevier Science. Pnel modified, with permission, from REF. 8 (2003) Elsevier Science. PATCH-CLAMP CAPACITANCE TECHNIQUE A glss pipette is seled ginst the memrne nd n lternting voltge signl pplied. The induced current is recorded nd used to clculte the memrne cpcitnce. The cpcitnce of the memrne is proportionl to its surfce, nd so gives mesure of the mount of exocytosis or endocytosis tking plce. concern in interpreting dt from mmmlin systems is tht they re often investigted t room temperture: switching to physiologicl temperture ffects oth exocytosis nd endocytosis of vesicles 148,149. In goldfish ipolr cells, it hs een shown tht slow endocytic process (time constnt ~10 s) nd fst process (time constnt ~1.25 s) coexist 12.A morphologicl correlte to the two pthwys ws indicted y the experiments in REF. 129, which showed tht exocytosis ws followed y ulk memrne uptke (slow endocytosis?) nd possily lso single vesicle uptke (fst endocytosis?). Formtion of vesicles from infoldings fter stimultion through clthrin-cot mechnisms hs lso een suggested in different rion synpse (frog scculr hir cells 150 ). Both endocytic processes seem to refill the reserve 72. In conclusion, it cn e proposed tht recycling vesicles re generlly retrieved through endocytosis directly from the plsm memrne, wheres reserve relese is followed y ulk endocytosis. One interprettion is tht molecules in recycling vesicle memrnes re glued tightly together, nd the vesicle memrne is continuous fter exocytosis. The fused vesicle memrne ptch might e locted y the endocytic mchinery through interctions etween dptor proteins nd integrl vesicle memrne proteins By contrst, if the vesicle components spred into the plne of the memrne fter fusion 156,ulk endocytosis 72,125,129 might e the only solution to retrieving them. Sorting of the vesiculr components might occur during (or efore) clthrin-medited vesicle formtion from the infoldings. However, it should e noted tht ulk endocytosis is clssiclly viewed s n emergency response tht opertes when the norml endocytic routes re overwhelmed y intense relese 87,which is n entirely plusile interprettion. Kiss-nd-run recycling. A frction of the vesicles in mmmlin systems might recycle through n ultrfst endocytic pthwy, known s kiss-nd-run 155, in which vesicles fuse trnsiently to the plsm memrne, nd reform y the simple closure of fusion pore. Dt in support of this model hve een otined in hippocmpl outons using differentil relese from vesicles of dyes with different hydrophoicities 130,156, incomplete relese of dye from single vesicles on exocytosis 44 nd rpid endocytosis monitored y fluorescence 49.In clyx of Held synpses nd isolted pituitry nerve terminls, rpid endocytosis ws oserved using the PATCH-CLAMP CAPACITANCE TECHNIQUE 157,158.However,only frction of the 48 or recycling 148 vesicles use this pthwy. Prt of the intuitive ppel of very rpid endocytosis is the possiility of fst vesicle recycling, which could provide n importnt selective dvntge in synptic function. However, lthough kiss-nd-run dt indicte tht endocytosis is fst (su-second in some cses), the only mesure of complete recycling indictes tht s re needed for the vesicle to ecome relese-competent gin 44 not much fster thn estimtes for vesicles tht recycle into the recycling through the clthrinmedited route 8,37. Virtully no evidence for kiss-nd-run endocytosis hs een otined in goldfish ipolr cells 75,154 or t the frog NMJ 8,24,37,ut in Drosophil evidence of kiss-ndrun endocytosis hs een climed for knockouts of endophilin protein tht is involved in endocytosis 159. Moreover, electrophysiologicl recordings (FIG. 7c) showed tht the synptic rundown in shiire mutnts t high temperture is very rpid 9.Ordwy nd collortors 160 nlysed synptic depression in Drosophil with higher time-resolution, nd found tht the relese from shiire neurons t the non-permissive temperture ws 66 JANUARY 2005 VOLUME 6