Neurosc ienrr Vol. pp. I773 to I804 Pergamon Press Lid 1979. Prmted m Great Brilam OlBRO

0306.4522’79’1201.1773SO2.00,0

COMMENTARY’ VESICLE RECYCLING

AND TRANSMITTER

RELEASE

H. ZIMMERMANN Max-Planck-Institut fur Biophysikalische Chemie. Abt. Neurochemie, D-3400 Gattingen-Nikolausberg. Federal Republic of Germany

The development of a concept Consequences of the vesicle hypothesis Vesicle recycling established by fine-structural methods Vesicle depletion and replenishment Extracellular markers Specificity of marker uptake Fate of vesicles Different types of vesicles and cisternae Freeze fracture Relation of released transmitter to vesicle-bound transmitter Preferential release of newly synthetized transmitter The difficulty in defining subcellular compartments Metabolic studies using tissue fractionation methods Vesicle heterogeneity revealed by density gradient centrifugation studies Cholinergic systems Adrenergic systems Application of false transmitters Relation of vesicle recycling and reloading to turnover of transmitter Vesicular uptake Nerve terminal uptake The postulate of stoichiometric release Recycling of vesicle membrane Membrane constituents Fusion and retrieval Quantum hypothesis and vesicle hypothesis Correlation between statistical parameters and function Changes in quanta1 size and statistical parameters Is the quantum quantized? Summary and conclusions

THE STUDY of synaptic transmission has fascinated investigators for many decades since it offers a cell biological problem which can be approached by a number of different techniques simultaneously. Lightand electronmicroscopy reveal the transmitting structures, electrophysiology monitors the changes in potential across the cell membranes and their dynamic behaviour, and finally biochemistry attempts an understanding of the phenomena at a molecular level. Thus it becomes clear that the study of synaptic transmission cannot be restricted to any of these technical fields and any statement which is based on a single technical approach is necessarily incomplete. The main object of this commentary will be the t This commentary is dedicated to Dr V. P. Whittaker on the occasion of his 60th birthday. Abbreuiarions: ACh, acetylcholine; ATP, adenosine S-triphosphate; EDTA, ethylenediamine tetra-acetate; m.e.p.p., miniature endplate potential. MC.

4112-A

question whether vesicle recycling and transmitter release are immediately connected, i.e. whether they relate to an identical cellular process. There will be little doubt that synaptic vesicles recycle during induced transmitter release, but the difficult question to answer is whether these vesicles also release and recycle the transmitter or whether recycling vesicles might be involved in functions other than transmitter release. In the case of (conventional) synapses the released substances are rather small molecules which can be rapidly synthesized within the nerve ending and for which (or for whose degradation products) there will be rapid mechanisms for reuptake and reutilization. In the case of neurosecretory systems the active substances are synthesized and packaged in the cell body and transported to the nerve ending inside storage granules where they are finally secreted into the body fluids. It is therefore possible that there exist differ-

1773

I773

H. ZIMMERMANN

ences in the molecular mechanisms involved in storage and release in the various systems. A number of reviews have appeared in which various aspects of transmitter release or vesicle recycling have been discussed, some of which will provide the reader with more detailed references on specific points (KKNJFVI~.. 1974: FIN~YSON & OSRORNF,.

vesicle was originally estimated to be 100&2000 mols (WHITTAKER& SHERIDAN, 1965). This compares well with the ACh content of cholinergic vesicles isolated from bovine superior cervical ganglion (1600 mols; WILSON. SCHULZ SCCOOPER,1973). The larger vesicles of the Torpedo electric organ may contain as many as 200,000 mols per vesicle (OHSAWA, I&WE, MORRIS & 1975: BARKER. 1976: BARRI:T & MAGLERY, 1976; WHITTAKER, 1979) and those of Narcine 50,000 HANIN & COSTA, 1976; MCINTOSH & COLLIER,1976; (WAGNER, CARLSON & KELLY. 1978). Apart from the HOI,TZMAN, 1977: HOLTZMAN. S~HACHER, EVANS & higher values for large vesicles isolated from electric TEICHRERG.1977; TRIFARb. 1977: LL.IN,~S& HEUSER. tissue the estimated number of molecules is of the 1978: MELDOLES],BORGESE.DI: CAMILLI & CE Mauthner fiber--giant fiber synapse. hatchet fish Synaptosomes Rat cerebral cortex Torpedo electric organ

Noradrenaline GABA

GABA, glutamate

Unknown

& MAURO. 1972); GABA,

y-aminobutyric acid

FRIED & BLAUSTEIN(1978) MICHAELSON.BILEN & VOLSKY (1978)

High K +. veratridine Ca’ + ionophor CLARK, Hu~Lm:t

MODEL. HIGHSTEIN& BENNET(1975)

Electrical

The rable does not include reports on vesicle depletion with spider venoms (e.g.

COOKE. CAMERON& JONES(1975) FEAR, Joi, & HAL&Z (1972)

REINECKE & WALTHFB(1978) ATWOOD,LANG & MORIN (1972)

PERRI, SACCHI, RAVIOLA& RAVIOLA(I972) BASBAUMKc HEUSER(1979) BOTHAM,BEADLE,HART. POTTER& WILSON(1978):

(1972, 1974); BILKS(1974)

DE ROBERTIS& VAZ FERREIRA(1957) PARDUCZ & FEHER(1970); FRIESEN& KHATTER(1971); PYSH & WILEY

HUBBARD & KWANHUNRUMPEN (1968) JONES& KWANBUNBUMPEN(19700); K~RNF~ IUSSIX (1972) HEWER & M~LEIX (1971) CECCARELLI,HURLBUT & MAURO (1972, 1973); HEUSER& REESE(1973) GENNARO. NASTUK & RUTHERFORD(1978) ZIMMERUANN& WHITTAKER(1974a, h); BoYNE, B~HAN & WILLIAMS(1975)

Reference

High K’ Acoustic stimulation

Electrical, in the presence of dinitrophenol

Electrical Electrical

Rat superior cervical ganglion Mouse vas defetens Locust motor nerve

High K+ Electrical

Stimulus

SYNAPTICVESICLES IN VARIOUSTRANSMITTER SYSTEMS

High K’ Electrical La” (1 mM) Electrical

DEPLETION

Electrical Electrical

Narrine)

junction

Electric organ (Torpedo.

Frog neuromu~nlar

Neuromuscular junction Rat diaphragm

Tissue

1. ST~IULATION

Preganglionic sympathetic neuron Rabbit splanchnic nerve, adrenal medulla Cat superior cervical ganglion

Acetylcholine

Transmitter

TABLE

HRP, horseradish peroxidase.

Noradrenaline GABA, glutamate Peptides (neurosecretory)

Acetylcholine

Transmitter

Median eminence Central nervous system in oiw Rabbit, cat, monkey cerebral cortex Rat cerebral cortex Total rat brain Central nervous system in vitro Rat cerebral cortex Spinal cord explant Synaptosomes Rat cerebral cortex Retina Frog, skate, turtle

Mouse vas deferens Lobster neuromuscular junction Rat, hamster pituitary

Torpedo electric organ

Frog neuromuscular junction

Tissue

COOKE, CAMERON& JONES(1975) TEICHEIERG, HOLTZMAN, GRAIN & PE?ERSON(1975) FRIED & BLAUSTEIN(1976, 1978) SCHACHER,HOLTZMAN & HOOD(1974, RIPP~, SKAHIB & MCDONALD (1976); SCHAEFFER& RAVIOLA (1978)

HRP HRP HRP, thorotrast HRP

1976);

TURNER & HARRIS (1973, 1974) JONES,CAMERON& ELLI~~N (1977) JBRGENSON& MELLERUP(1974)

CECCARELLI,HURLBUT & MAURO (1972, 1973); HEUSER& REESE(1973); HURLBUT & CECCARELLI(1974) POLITOFF,BLITZ & ROSE(1975) PREVITE.ROSE, BLITZ & POLITOFF(1976) HEUSER& LENNON(1973) ZIMMERMANN& DENSTON(1977~) BA~BAUM& HEUSER(1979) HOLTZMAN, FREEMAN& KASHNER(1971) NAGASAWA, DOUGLAS & SCHULZ (1971); THEODOSI$DREIFUSS,HARRIS & ORCI (1976) NORDMANN, DREIFUSS,BAKER, RAVAZZOLA, MALAISSE-LAGAE& ORCI (1974) PELLETIER,DWQNT & PWIANI (1975)

Reference

HRP HRP Albumin

HRP

HRP, mannitol. inulin, albumin

Acetylcholinesterase ATPase (apyrase) HRP Dextran HRP HRP HRP

HRP, dextran

Extracellular marker

TABLE 2. UPTAKE OF EXTRACELLULAR MARKERSINTO SYNAPTICVESICLES OF VARIOUSTRANSMITTER TYPESIN DEPENDENCE OF NERVEACTIVITY

17x0

H. ZIMMERMAN~

In a recent study on the sympathetic neurons innervating the mouse vas deferens a stimulation-dependent fall in vesicle numbers was observed selectively in the small vesicle population whereas the (few) large vesicles appeared not to be affected. Replenishment of the small vesicle population was almost complete after 2 h of recovery (BASBAUM& HEUSER, 1979). Preferential depletion of the small vesicles has also been noted after the application of red back spider venom (HAMIL.TON& RORINSON.1973). Eutrucellular

markers

A more direct demonstration of vesicle recycling is achieved by the application of extracellular markers which cannot pass the external presynaptic membrane. Such high molecular weight compounds like the protein horseradish peroxidase or the polysaccharide dextran are taken up into synaptic vesicles of activated nerve endings from various tissues including neuromuscular junctions spinal cord explants or retinae but also into synaptic vesicles of ‘resting’ brain tissue in rino (Table 2). Even extracellularly applied acetylcholinesterase (POLITOFF, BLITZ & ROSE, 1975) or adenosine-S-triphosphatase (ATPase, apyrase) (PREVITE,ROSE, BLITZ & POLITOFF, 1976) are taken up into cholinergic nerve endings and subsequently block synaptic transmission. However apyrase in addition causes vesicle depletion. Finally, with horseradish peroxidase and thorium dioxide it was shown that synaptic vesicle recycling can still occur in isolated synaptosomes (FRIED & BLAUSTEIN, 1976: 1978). Because synaptosomes are derived from a variety of terminals this work provides evidence that vesicle recycling occurs in all transmitter systems of the brain. It should be stressed that under conditions of sustained low frequency stimulation where there is no loss of vesicle counts, the participation of an increasing number of synaptic vesicles in exo- and endocytosis can only be observed by incorporation of extracellular marker (HURLBUT & CECCARELLI,1974; CECCARELLI& HURLBUT, 1975; ZIMMERMANN& DENSTOP*‘, 1977~). Blockage of choline uptake by hemicholinium-3 under these conditions of stimulation does not impair vesicle recycling in the frog neuromuscular junction (CECCARELLI& HURLBUT, 1975) but results in depletion of the presynaptic stores of transmitter as measured by the disappearance of quanta. In the Torpedo electric organ a progressive depletion of the content of both ACh and ATP in recycling synaptic vesicles (in the absence of hemicholinium-3) can also be observed directly (ZIMMERMANN& DENSTON, 19776). These results suggest that vesicle recycling and the repletion of vesicle stores of transmitter are two independent processes. In neurosecretory terminals in the pituitary gland, horseradish peroxidase is taken up into vesicular structures when hormone release is induced. Both the preferential uptake of the enzyme into small (microvesicles (NAGASAWA, DOUGLAS & pinocytotic) SCHULZ. 1971) and into vacuoles of larger diameter

(NORDMANN,DREIFUSS,BAKER,RAVAZZOLA,MALAISSELAGAE& ORCI, 1974; THEODOSIS,DREIFUS$ HARRIS & ORCI. 1976) have been described. Possibly the intensity of the secretory stimulus influences the type of endocytotic route which is used for membrane retrieval. In peripheral adrenergic nerve varicosities the uptake of horseradish peroxidase was found to be restricted to small vesicles which in diameter correspond to the small dense-cored vesicle population (BASBAUM& HEUSER. 1979). Specijcity

qf murker

uptukr

Since markers like horseradish peroxidase are taken up into endocytotic vesicles not only in other secretory systems (for references see HOLTZMAN, 1977; MELDOLESIet al., 1978) but also may be taken up all over the surface of a nerve cell (TURNER & HARRIS 1974; TEICHBERGet cd., 1975) or even on sensory neurites (CHOLJCHKOV,1974; WELDON, 1975) it may be argued that the uptake of these substances is nonspecific. The experiments on the nerve endings, however, clearly demonstrate that there is only significant uptake of marker during induced transmitter release (e.g. TEICHBERGet cd.. 1975; CECCARELLI& HURLBUT, 1975; ZIMMERMANN& DENSTON, 1977~; FRIED & BLAUSTEIN,1978). Spontaneous release of quanta does not appear to cause significant alterations in vesicle morphology. The fact that the extracellular marker is taken up into synaptic vesicles when a physiological stimulus is applied makes it possible to use this technical approach in turn to monitor the activity of a synapse in intact nervous tissue (SCHACHER ef al., 1976: EVANS, HOOD & HOLTZMAN. 1978). Extracellular marker is taken up into vesicles of retinal receptor cells during darkness only. This process is inhibited by illumination, as would be expected from previous physiological studies (see also RIPPS er ul.. 1976: SCHAEFFER& RAVIOLA, 1978). It appears, however. that there are substances whose uptake into nerve endings is mediated by meshanisms which are not immediately related to synaptic activity. This has become obvious from studies on retrograde axonal transport. Generally, high molecular weight substances like horseradish peroxidase can be taken up into the axon terminal and transported in a retrograde manner to the perikaryon (e.g. KRISTENSSON. OLWN & SJOSTRAND. 1971; MALMGREN, OLSSCIN,OLS~ON & KRISTENSSON. 1978). The amount of material transported via this mechanism is, however, very small unless the substance possesses a special affinity for the nerve terminal membrane. Certain molecules like nerve growth factor, tetanus toxin, antibodies to dopamine fl-hydroxylase, cholera toxin and several lectins can be taken up into the nerve endings with high selectivity and become transported back to the perikaryon (DUMAS, SCHWAB & THOENEN. 1979). As compared to other macromolecules like horseradish peroxidase.

Vesicle recycling and transmitter release

1781

reticulum system (DROZ, RAMBOURG& KOENIG,1975). Substances are transported in a retrograde manner and this transport occurs inside smooth is several hundred times less (ERDMANN,WIECAND & vesicles, tubes or cisternae; they may be degraded in WELLH~~NER, 1975; PRICE, GRLFFIN,YOUNG, PECK & STOCKS 1975; STOECKEL,PARAVICINI& THOENEN, multivesicular bodies or lysosomes (e.g. NAUTA, KAISERMAN-ABRAMOF 8~ LASEK, 1975; TEICHBERGet al., 1974; STOECKEL, SCHWAB & THOENEN,1975a,b; 1977; 1975; LAVAIL 8~ LAVAIL, 1975; BROWNSON, UUSITALO FILLENZ, GAGNON, STOECKEL& THOENEN, 1976; & PALKAMA,1977; TURNER,1977). On the other hand STOECKEL,GUROFF, SCHWAB & THOENEN, 1976; PRICE, GRIFFIN & PECK, 1977; SCHWAB & THOENEN, the vesicle-bound extracellular marker can reside inside the nerve terminal for several hours (TEICHBERG 1978; SCHWAB,JAVOY-AGID& AGID, 1978). The speet al., 1975) and the experiments of HEUSER& REESE cial characteristic of these substances is obviously (1973) and CECCARELLI& HURLBUT (1975) suggest their ability to bind with a high affinity to specific that it can be released on renewed nerve stimulation. components of the nerve terminal membranes. These Thus the possibility that part of the marker-loaded include receptors for nerve growth factor, dopamine membrane structure will undergo retrograde transfl-hydroxylase for the respective antibody, monosialoport and final degradation (see also HOLTZMAN,1977) gangliosides for chplera toxin, di- and trisialogangliodoes not exclude the possibility that the majority of sides for tetanus toxin and various sugar moieties of the vesicles will continue to recycle at the nerve endcell surface glycoproteins for lectins. ing. By analogy, anterograde transport as a means of A further major difference from the group of subsupplying transmitter substances like noradrenaline stances mentioned above appears to be the fact that for horseradish peroxidase and similar substances sig- or ACh appears to be negligible compared to the nificant uptake is only achieved under conditions of amounts in the terminals or their turnover (for references see TUEEK, 1978). induced transmitter release, whereas it has been shown at least for nerve growth factor (STOECKEL, DUES & THOENEN,1978) that its uptake and subDifferent types of vesicles and cisternae sequent retrograde transport is not influenced by Vesicles may differ in other ways than their content neuronal activity. In the rat visual system, specific of marker. Coated vesicles which are obviously condifferences were observed in the behaviour of various isoenzymes of horseradish peroxidase (BUNT & nected to pinocytotic activity in various tissues have also been observed in the activated synapse. The presHASCHKE, 1978); those which did not undergo ence of coated vesicles in the fine-structural picture obvious retrograde transport were found to be confined to synaptic vesicles and did not enter other seems, however, to be dependent on the fixation membrane compartments of the nerve terminal. method used (GRAY, 1972; 1975a; PAULA-BARBOSA & It is concluded that the basis for nonspecific uptake GRAY, 1974; PAULA-BARBO.SA, SOBRINHO-S&ES & at the nerve ending is the trapping of molecules as a GRAY, 1977). Thus it is possible that the presence or result of transmitter liberation by exocytosis and subabsence of coated vesicles in the nerve ending sequent membrane retrieval. The nonspecific nature (HEUSER& REESE,1973; CECCARELLI et al., 1973); choof this process would explain the high concentration linergic terminals) cannot be used as a strong arguof marker necessary to obtain uptake of measurable ment in the discussion of the membrane retrieval proamounts. cess. Coated vesicles do not appear to be involved in the vesicle retrieval process at the adrenergic nerve terminals of the vas deferens (BASBAUM& HEUSER, Fate of vesicles 1979). The question arises whether synaptic vesicles which In a number of cases a decrease in vesicle diameter have taken up extracellular marker could be involved was observed on stimulation (JONES& KWANBUNin physiological processes other than transmitter storBUMPEN,1970a; KORNELIUSSEN, 1912; MCKINLAY & age and release. Morphological studies on the fate of USHERWOOD,1973; MALOSHEVSKI, LEINKOV& BRAsuch vesicles could help to answer this. On sustained GINA, 1976). This is particularly apparent in the Torstimulation at low frequency (which does not deplete pedo electric organ (ZIMMERMANN& WHITTAKER, the number of vesicles) 50% or more of the entire 1974~; ZIMMERMANN& DENSTON, 1977a) where the vesicle population may become labelled (HURLBUT& vesicles re-formed on stimulation are about 25% CECCARELLI,1974; CECCARELLI& HURLBUT, 1975; smaller than the original ones. Clusters of these small ZIMMERMANN & DENSTON,1977a). Although it seems vesicles are found in close apposition to the presynapunlikely that the ‘true’ (transmitter-containing) vesicle tic membrane (ZIMMERMANN,1979a). Furthermore, population during activation of the synapse will this population was shown to be the one which conbecome replaced by a vesicle population with a differtains the extracellular marker. In contrast FRED & ent function, the possibility that there are different BLAUSTEIN (1978), using isolated synaptosomes during types of synaptic vesicles inside a nerve ending has to very brief recovery periods, observed an initial uptake be faced. of marker into larger vesicles which later appear to be Synaptic vesicles may be connected to a general reprocessed to form smaller ones. Here too, the albumin or ferritin the amount of protein that needs to be applied in order to detect retrograde transport

axoplasmic

details of the fine-structural picture might well vary with the choice of fixation and experimental conditions. Similarly the extent to which reticular systems 01 cisternae appear to be involved in vesicle recycling (HEUSER & REESE, 1973: MODEL c’t trl.. 1975) or not (CECCARELLIYI ul.. 1973; ZIM~RMANN & DENSTON. 1977~) (compare Fig. I) may be a result of the fixation conditions. Furthermore. structures appearing as cisternae on thin sections may turn out to be infoldings when serial sectioning is applied (ROSE. PAPPAS & KRIEREL. 1978). There could. however. be another explanation for the contradictory results concerning the recycling of vesicles via cisternae. The involvement of tubular or reticular systems is generally observed when a massive fusion of vesicles had been induced previously. If. however. more physiological conditions of stimulation are applied which do not decrease the number of vesicles. the fine-structural picture of the synapse is very much like that of a control. Vesicles may, however, contain extracellular marker (e.g. CEWARELLI & HURLWIT. 1975: ZIMMERMANN & DENSTON. 1977~: SCHAEFFER & RAVK)L.A. 1978). It is likely that involvement of cisternae to a greater extent in vesicle recycling occurs only if the outer cell surface has been extensively enlarged experimentally and that it does not occur under conditions where vesicle recycling can keep pace with vesicle exocytosis. This would also explain the reproducible appearance of the presynaptic fine structure when samples are taken from living (central or peripheral) nervous tissue. It is concluded that synaptic vesicles will start to recycle in a nerve ending when it is stimulated. Using high concentrations of nonspecific extracellular marker this will be taken up into the vesicle lumen. Although the possibility for retrograde transport exists, most vesicles are capable of renewed recycling. This process is not restricted to peripheral synapses but occurs also in central nervous tissue and may occur equally well in all transmitter systems. The results of studies on the specific uptake of a number of substances by non-activated nerve terminals suggest that the nerve ending and its membrane compartments are also able to serve additional cellular processes. There is now a need for biochemical methods which would allow the separation of vesicular or reticular systems of different functional states in order to elucidate their role in the nerve ending.

Freezefracture

AKERT &

LANDIS, 1974;

PEPER, DREYER, SANDRI, AKER~ &

MOOR. 1974; HEUSER, 1976). The number of small dimples or vesicle attachment sites on the inner leaflet of the external presynaptic membrane which are interprcted as sites of vesicle fusion depends on the activity of the synapse and increases on stimulation (STREIT. AKERT. SANDRI, LIVINGSTON& MOOR. 1972; HCUSER c’f 01.. 1974: PFENNINGER& ROVAINEN. 1974; CEKARI-1.1.1.GROHOVAI, HURLHUT & JEZZI. lY7Ya. h). Simi-

larly. conditions which induce release of neurohypophysial hormone will result in an increase of exocytotic profiles in neurosecretory terminals (THEOIX)SIS. BIJRLET. BOIJDIER& DREIFUSS,1078).

A rather detailed picture of the exocytotic process has been obtained at the frog neuromuscular junction Losing two different experimental approaches. The one involves extremely rapid freezing of the tissue (HEUSER. REESE& LANDIS, 1976) during activation of the synapse in the presence of 4-aminopyridine (HWSER, 1977: LI.IN~S & HHJSER. 1978; HEUSER. RwsI-, DENNIS. JAK. JAN & EVANS. 1979). The other one uses the intensified discharge due to black widow spider venom which is compared to the effect of depolarization by excess K ’ (CECCARELLIrt ~1.. lY7Y0.b). From both studies it becomes clear that the sites of vesicle fusion are the active zones described pre\iously in electron-microscopic studies on thin sections (Cor~rr~l:x. 1974). Even if the ridges of the active zone which are typically bordered by a parallel row of membrane-bound particles are heavily distorted by addition of a calcium chelating agent, the resulting fusions induced h> black widow spider venom always appear strictly adjacent to the rccopnizable remnants of the double rows (CE(.CARELI.I. HtIRIX’T. DE CAMII.I.I & MELWLESI, 1978: O:C‘