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The subcellular origin of the agetylcholine released at synapses
Int. J, Biochetn., 1975, Vol. 6, pfi+ 303 to 312. Pergamon Press. Printed in Great Britain
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MINIREVIEW THE SU~CELLULAR
ORIGIN
RELEASED
OF THE ACETYLCHOLINE
AT SYNAPSES
R. M. MARCHBANKS Department of Biochemistry, Institute of Psychiatry, London SE5 8AF, U.K. (Received23 October 1974)
SHORTLY after the enunciation of the quanta1 theory of synaptic transmission by Del Castillo & Katz in 1954, small vesicles were observed in the synaptic region by electron microscopy (De Robertis & Bennett, 1955). It seemed reasonable, as suggested by Del Castillo & Katz (1955) that these vesicles were the morphological counterpart of the quanta and it was envisaged that they could quantitatively release their contents into the synaptic cleft by reverse micropinocytosis or exocytosis (Del Castillo & Katz, 1957). The demonstration by Whittaker, Michaelson & Kirkland ( I 964) that these vesicles did indeed contain the neurotransmitter acetylcholine completed the triad of observations, physiological, morphological and biochemical, the unification of which into a theory of transmitter storage and release is proposed by the vesicular hypothesis. Despite considerable attention from investigators for 20 years there remain difficulties with the vesicular hypothesis. In this short review an attempt wiI1 be made to assess its current status. The vesicular hypothesis has a weak and a strong form. The former merely states that the vesicles store the neurohumoral agent and must therefore release it somehow for the purpose of chemical transmission. The strong form, with which I am concerned here, states that the vesicles store a defined quantity of transmitter (the quantum) and release it quantitatively outside the terminal during the process of transmission. Although other mechanisms have been proposed, the most favoured is of acetylcholine release by fusion of the vesicle with the pre-terminal membrane and total exocytosis of the
vesicular content, This proposal has become an integral part of the strong form of the vesicular hypothesis and it is worth noting that it was inspired in a morphological tradition to explain a physiological phenomenon, but its proof or disproof must rely on biochemical arguments. It is necessary to show that the acetylcholine released on stimulation has come directly from within the vesicle without passing through the cytoplasm. PHYSIOLOGICAL EVIDENCE There have been three different approaches to determine the amount of acetylcholine in a quantum and they have given figures for the number of acetylcholine molecules ranging over three orders of magnitude. (I) Direct comparisons between the amount of acetylcholine released and the number of impulses divided by the number of end plates and the number of quanta per impulse have been reported by several authors (McIntosh, 1959; Straughan, 1960, KrnjeviC & Mitchell, 196x). The values range between 498 and ~oo,ooo molecules of acetylcholine per quanta. Some of the discrepancies are considered by Hubbard (1970) and a preferred figure of 40,000 emerges. (2) A comparison can be made of the content of releasable acetylcholine in the nerve terminal and the number of quanta released by stimulation to exhaustion when synthesis of acetylcholine is blocked. This procedure yields a value of 57,000 molecules of acetylcholine per quanta (Elmquist & Quastel, r 965). (3) Attempts to mimic the miniature end
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plate potential by electrophoretic application of acetylcholine suggest that 100,000 molecules of acetylcholine are needed (Miledi, 196 I) but presumably this is an upper limit because the injecting micropipette cannot be placed as advantageously as the site of release of acetylcholine. Somewhat different results have been obtained by the analysis of acetylcholineinduced membrane noise (Katz & Miledi, 1972). It is assumed that the noise arises from random variations in the rate of molecular collision between molecules of acetylcholine and receptor on the postsynaptic membrane. Each successful collision produces a voltage change which contributes to the amplitude of the acetylcholineinduced membrane noise measured as the variance of the membrane potential. Making certain corrections for non-linear summation, Katz & Miledi (1972) were able to calculate the characteristics of the elementary acetylcholine current pulse. Each collision of a single acetylcholine molecule with the receptor produced a conductance change of 10-10 mhos lasting for 10-3 seconds, which caused a depolarization of 0.3 PV and was about 1-2000 times smaller than the miniature end plate potential. In a further study, Katz & Miledi (1973) estimate that about 60% of the molecules of acetylcholine that are released into the synaptic cleft are bound to receptor. They conclude that about 30,000 molecules (i.e. 60% of the 50,000 molecules in the quantum estimated as described previously) are bound to receptor but that only 5-1oO/~ of the acetylcholine-receptor complex is in the active form that allows the passage of ions across the membrane. This supposes a very ‘ inefficient ’ acetylcholine receptor and an alternativeexplanationisthattheacetylcholine receptor complex approaches a reasonable efficiency but that the figure of 50,000 molecules of acetylcholine per quantum is a gross overestimate. This could be because a large amount of acetylcholine is released on stimulation in a non-quanta1 fashion but is attributed to quanta in the calculation, In fact thus causing the overestimate. 50,000 molecules can only be packed inside
Int. J. Biochem.
a vesicle if the acetylcholine is crystalline (Canepa, I 964) and the biochemical evidence (see below) suggests that mammalian synaptic vesicles contain much less acetylcholine than this. These uncertainties make it difficult to try and define a mechanism of release the physiological which would satisfy observations. BIOCHEMICAL EVIDENCE The isolation of synaptic vesicles and the demonstration that they contain acetylcholine (Whittaker et al., 1964) is probably the single most important result of biochemical studies on the topic. Without this demonstration the role ascribed to the vesicles in chemical transmission would be largely hypothetical. Vesicles from the cholinergic synapses have been isolated from three tissues; cerebral cortex of laboratory rodents, electric organ of Torpedo marmorata and from bovine superior cervical ganglion. In each case the acetylcholine content of the individual vesicles has been estimated by adding a known amount of polystyrene latex beads to the preparation so that the ratio of beads to vesicles in the field of an electron micrograph can be used to calculate the number of vesicles in the preparation and hence the ratio of acetylcholine molecules to vesicles. Synaptic vesicles from mammalian tissues contain between IOOO and 2000 molecules of acetylcholine (Whittaker & Sheridan, 1965; Wilson, Schulz & Cooper, 1973) while those from Torpedo electric organ contain rather more (Whittaker, Essman & Dowe, ‘972). The vesicles lose their acetylcholine if submitted to hypo-osmotic conditions and this is most conveniently demonstrated by gel-filtration in solutions of different osmolarity (Marchbanks, 1967; Marchbanks & Israel, rg7 I). One may conclude from this that the acetylcholine is sequestered by a semi-permeable membrane and is therefore within the vesicle rather than merely absorbed to the surface. If the concentration of acetylcholine inside the vesicle is calculated it is found to be rather high, ranging about iso-tonicity. Information about how the acetylcholine
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is taken up and retained within the vesicle is incomplete. A study of the association of recently synthesized acetylcholine with synaptic vesicles has offered some clues to the nature of the process. When radioactive acetylcholine is synthesized from labelled precursor in a closed system the more recently synthesized stores will have a higher specific radioactivity. It was noticed (Marchbanks & Israel, 197 r) that the acetylcholine of vesicles isolated from tissue incubated with labelled precursor displayed heterogeneity on gel-filtration. The recently synthesized acetylcholine of high specific radioactivity was lost preferentially from the vesicles when they were passed over iso-osmotically eluted Sephadex columns, whereas the non-radioactive preformed acetylcholine was retained. Using the technique of gel filtration in this way it was possible to distinguish the loosely bound recently synthesized store from the tightly bound preformed store of acetylcholine and to compare their properties (Marchbanks & Israel, 1973). It was found that the loosely bound acetylcholine could become tightly bound under various conditions but the extent of the conversion to the tightly bound form was always positively correlated with losses from the original tightly bound form. For example, incubating at pH 5 or with calcium ions increased the incorporation of radioactive acetylcholine from the loosely bound store, but at the same time losses from the tightly bound store were increased. Incubation at 6” C or 20’ C produced comparatively little change in either store. Furthermore, factors at present unknown that positively affected exchange from the loosely bound store into the tightly bound store positively affected exchange out of the tightly bound store with high statistical significance (Marchbanks & Israel, I 972). The most parsimonious explanation is that the exchanges in each direction are coupled. The coupling of the exchange between recently synthesized loosely bound acetylcholine and the preformed store of tightly bound vesicular acetylcholine must imply that both stores exist in the same vesicle, because free acetylcholine could not diffuse
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between vesicles without being hydrolyzed by residual cholinesterase present in the preparation. The results are best explained by postulating that the recently synthesized loosely bound acetylcholine exists on or near the surface of the vesicle and that it cannot exchange into the tightly bound form without the vacation of binding sites in the core of the vesicle by the pre-formed and hitherto tightly bound acetylcholine. No association of the loosely bound acetylcholine with membrane fragments could be discerned. A study of the relationship between ATP (Marchbanks & Israel, 1972) and acetylcholine of vesicles (Dowdall & Zimmermann, 1974) also suggests two vesicular pools of the transmitter, one identified as being labile, rapidly lost when the electric organ is stimulated, having little associated ATP and being possibly similar to the loosely bound compartment, while the other is more stable and has an acetylcholine: ATP molar ratio of about 4 : I. Quantitatively the rate of exchange between compartments is small, so that pre-labelled vesicles are required to demonstrate it. It is probable that the action of calcium ions in hastening the rate of exchange and hence release of acetylcholine from the vesicles is of importance in the mechanisms of synaptic transmission. Stimulation of the electric organ results in entry of calcium into the pre-synaptic tissues and the progressive increase of calcium concentration is accompanied by a progressive decrease in the bound (vesicular) acetylcholine (Babel-Guerin, I 974). It seems likely that there is no hard and fast distinction between the loosely bound and tightly bound store: the acetylcholine of each concentric shell progressing inwards towards the core of the vesicle is progressively more tightly bound. Conversely progressing outwards, the acetylcholine of each concentric shell is more and more labile and also the rate at which the equilibrium between it and the extra-vesicular acetylcholine is achieved becomes progressively more rapid. In order to explain the phenomena of the exchanges, the ability of vesicles to store acetylcholine and their osmotic sensitivity, the vesicle membrane would have to be more
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permeable to acetylcholine than to its counter-ions within the vesicle. Subcellular fractionation studies show that choline acetyltransferase is a soluble enzyme unattached to synaptic vesicles or other organelles. (Fonnum, I 967 ; Marchbanks & Israel, 1972). The site of synthesis of the transmitter is therefore in the cytoplasm. The relative rates of synthesis compared with storage in vesicles or disposal will define the size of the extra-vesicular pool ofacetylcholine. This pool comprises some 20-50% of the total, and is not derived from the breakdown of vesicles and release of their acetylcholine because in labelling experiments it always has a different specific radioactivity. Nor is it described by the ‘ surplus ’ acetylcholine Birks & Macintosh (196 I) because its presence can be demonstrated without the aid of anti-cholinesterases. The localization of the recently synthesized acetylcholinehasbeeninvestigated bystudying the subcellular disposition of radioactive acetylcholine after the tissue has been allowed to synthesize transmitter from labelled precursors. The results on isolated tissues (Marchbanks, rg6g a; Ritchie & Goldberg, 1970; Haga, 197 I ; Richter & Marchbanks, 1971, b; Marchbanks & Israel, 1971, 1972; Collier, Poon & Salechmoghaddam, 1972 ; Molenaar, Polak & Nickolson, 1973 ; Israel, Hirt & Mastour-Frachon, 1973; Israel & Tucek, 1974) can be summarized thus: the rate of incorporation into the vesicular compartment is usually less than, sometimes equal to but never greater than the rate into Experithe extra-vesicular compartment. ments of this kind with the tissue in situ have only been done on brain. Their interpretation is complicated by the permeability barriers presented to the entry of radiolabelled choline and the difficulty of identifying the period of peak incorporation after the pulse of labelled precursor. The turnover rate is so fast that the initial peak cannot easily be observed and the simplest explanation of the results of Sparf (1973) and Kewitz, Dross & Pleul (1974) would seem to be that the label in the extra-vesicular compartment of acetylcholine is diluted out faster with recently synthesized non-radioactive acetylcholine
ht.
3.
Biochem.
than is the label in the vesicular compartment, indicating acetylcholine synthesis outside the vesicles. These results suggest that, as would be expected from the localization of choline the most recently synacetyltransferase, thesized acetylcholine is extra-vesicular but that it can be transferred to the vesicular compartment quite rapidly under certain It is unlikely that empty circumstances. vesicles are filled with newly synthesized acetylcholine providing a small population of highly labelled vesicules whose radioactivity is swamped by a larger population of vesicles containing preformed unlabelled acetylcholine, because as explained previously, the newly synthesized acetylcholine, insofar as it exists in the vesicle fraction, appears to be on the surface of each vesicle. There is no discrete population of highly labelled vesicles that with present techniques can be isolated by subcellular fractionation. The studies of Birks & Macintosh (1961) on the superior cervical ganglion identified two compartments of acetylcholine available for release by stimulation. The ‘ readily contains about releasable ’ compartment 20% of the total releasable acetylcholine and this can be released on stimulation; the so-called ‘ reserve pool ’ contains about 80% of the total releasable acetylcholine, which is fed into the ‘ readily releasable ’ compartment, making acetylcholine available for release at a steady rate. The relationship between the readily releasable acetylcholine and the recently synthesized acetylcholine was first established by Collier & Macintosh (1969). The acetylcholine of the superior cervical ganglion was completely labelled by perfusion with [3H]choline-Locke solution; perfusion was then continued with unlabelled choline and the ganglion stimulated. The acetylcholine that was released had a lower specific activity than that remaining in the ganglion stores. It was concluded that the acetylcholine recently synthesized from the unlabelled choline was preferentially released by the stimulation. This implies that the reserve store exists in a cul-de-sac from the main path of acetylcholine throughput. This important result had also been
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ACETYLCHOLINE
suggested by studies on the evoked release of [3H]acetylcholine from the surface of the cerebral cortex (Marchbanks, 1969 b; Chakrin, Marchbanks, Mitchell & Whittaker, 1972). In addition, studies on the acetylcholine released from cerebral cortex slices by high potassium concentrations also suggested that it came from a newly synthesized compartment (Richter & Marchbanks, 1971 a; Molenaar et al., Ig73), although in interpretating this result one should not assume that potassium and electrical stimulation are exactly equivalent. The preferential release of newly synthesized acetylcholine on stimulation takes place in the diaphragm (Potter, 1970) and also in the electric organ of Torpedo (Dunant, Gautron, Israel, Lesbats & Manaranche, 1972). Splenic nerve stimulation releases newly synthesized noradrenaline from the spleen (Kopin, Breese, Krauss & Weise, 1968) and this suggests that the phenomenon, in addition to being a well established feature of cholinergic systems, is also a general characteristic: of neurohumoral transmission. It will be recalled from the previous section that the extra-vesicular acetylcholine is more recently synthesized than that within the vesicles. Therefore it follows that the vesicle population as isolated does not represent the immediate source of releasable acetylcholine, since to be released by exocytosis the recently synthesized acetylcholine would have to be present in the core of the vesicle, whereas it actually exists free and on the surface of the vesicle. Much more direct and striking evidence concerning the origin of the released acetylcholine comes from the studies of Dunant and his colleagues at the Salp&tri&-e Hospital (Dunant et al., These ‘972). investigations stimulated the electric organ of Torpedo either in situ or when cut in slices and superfused with physiological salines. They then immediately determined the ‘ free ’ and ‘ bound ’ acetylcholine that remained resistant to hydrolysis when the tissue was homogenized. The bound acetylcholine had previously been shown to be equivalent to vesicular acetylcholine in Torpedo, because there is no formation of
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synaptosomes and the specific radioactivity of acetylcholine in the bound and vesicular compartments were found to be the same (Marchbanks & Israel, 197 I). The immediate result of stimulation was a drop in the concentration of free acetylcholine; prolonged periods of stimulation eventually caused a drop in the bound acetylcholine. The electrophysiological response to stimulation was initially a 3oV depolarization but it decreased rather rapidly to about 50% within one hundred stimuli. Removal of calcium from the medium or addition of a high magnesium concentration prevented the drop in the concentration of free acetylcholine and also reduced the electrophysiological manifestation (see also Babel-Guerin, ‘974). In further studies (Dunant, Gautron, Israel, Lesbats & Manaranche, 1974) it was shown that the decrease in the electrophysiological response was exactly paralleled by the decrease in free acetylcholine over the course of the first IOO stimuli; subsequently the free acetylcholine concentration increased and this was associated with a plateau in the decrease of the electrophysiological response. The bound acetylcholine concentration did not change during these events, and experiments with [‘*C]choline showed that there was no increased exchange of acetylcholine, so that rapid emptying and refilling of the vesicles with acetylcholine can be ruled out. Further prolonged stimulation did then cause a decrease in the bound acetylcholine and changes in the number of vesicles observable in the synaptic region. The results of Zimmermann & Whittaker (1974 a) confirm those of Dunant et al. (1972) in showing that after prolonged stimulation ( > 500 stimuli) there was a loss of vesicular acetylcholine. However they did not measure extra-vesicular acetylcholine and they proposed that acetylcholine has been released by exocytosis. In their next paper (Zimmermann & Whittaker, 1974 b) findings are reported that are inconsistent with this suggestion (see below). In the adrenergic system, evidence for exocytosis is easier to obtain because the vesicles contain soluble protein such as
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dopamine-fl-hydroxylase and chromogranin A. On stimulation of the splenic nerve, protein appears in the perfusate along with noradrenaline and this is taken to indicate exocytosis, because it is difficult to see how a protein could be released by any other process (Smith, 1971). It is noteworthy that much more noradrenaline relative to dopamine-,%hydroxylase is found in the perfusate than in the vesicles, suggesting that exocytosis is not the only release process. Analogous evidence for exocytosis in the cholinergic system is more difficult to obtain, because, despite some claims (cf. Whittaker, it has not been established that ‘973L cholinergic vesicles specifically contain a unique soluble protein. The observation by Musick & Hubbard (1972) that proteins (unidentified and of unknown provenance) are released from the hemidiaphragm on stimulation is therefore difficult to interpret in this context. However cholinergic vesicles appear to contain ATP (Dowdall, Boyne & Whittaker, 1974) and the Ca2+-dependent release of ATP on stimulation of the diaphragm (Silinsky & Hubbard, 1973) is therefore of interest. As evidence for exocytosis, the release of ATP is less persuasive than the release of a specific vesicular protein, because the concentration in the cytoplasm is at least IO times higher (Dowdall et al., I 974) than in the vesicles. ATP release could not be detected when the superior cervical ganglion was stimulated (Kato, Katz & Collier, 1974). The biochemical evidence concerning the cholinergic synapse therefore not only does not support exocytosis but also suggests that the extra-vesicular acetylcholine is first and preferentially lost on stimulation. The theory of exocytosis could be preserved if there were a population of labile vesicles which were preferentially filled with acetylcholine and which preferentially released it on stimulation and which also were not stable enough to survive tissue homogenization. As already remarked there is no biochemical evidence for this proposal. The accuracy of the measurements do not warrant the statement that exocytosis never occurs but it does not contribute the majority of the acetylcholine
released on stimulation. A recent experiment by Taut, Hoffmann, Tsuji, Hinzen & Faille (1974) demonstrated that transmission could be abolished by the injection into the cell of Acetylcholine inside acetylcholinesterase. the vesicles is protected from hydrolysis by the enzyme so it follows that the extravesicular acetylcholine is responsible for the physiological response in this experiment. MORPHOLOGICAL
EVIDENCE
Despite the tradition within which the vesicular hypothesis was developed, the morphological evidence has always been rather scanty and contradictory. Until recently the event of exocytosis had only been rarely visualized and even then only in systems (unlike the cholinergic system) where the core of the vesicle is electron-dense (GrynszpanWinograd, 1971). What has been demonstrated with the aid of an electron-dense reaction product of horseradish peroxidase is pinocytosis at synapses (Holtzman, Freeman & Kashner, 1971; Heuser & Reese, 1973). The latter authors observe that on stimulation, reaction product is seen in coated vesicles formed at the edge of the synapse, presumably by pinocytosis. The coated vesicles fuse to form cisternae and after a period the reaction product is found in synaptic vesicles from whence it disappears during further stimulation. In addition, Heuser & Reese (1973) show that on stimulation, synaptic vesicles disappear and the terminal membrane gains (supposedly by exocytotic fusion of the vesicle) an equivalent amount of membrane which is subsequently transferred to the cisternae. However, Birks (1974) was not able to observe any expansion of the terminal area cervical ganglion on in the superior stimulation. The weakest link in this elegantly observed chain is that relating to the loss of horseradish peroxidase from the vesicles. It could have disappeared by mechanisms other than exocytosis, perhaps enzymic degradation, for example. Furthermore, even if the enzyme were expelled by exocytosis there is no reason to suppose that these particular vesicles contain acetylcholine or that the process is representative of those
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that do. It is worth recalling in this connection the synapses that are thought to be electrically transmitting and therefore presumably do not contain stores of transmitter are nevertheless well supplied with synaptic vesicles (Bennett, Pappas, Gimenez & Nakajima, rg67), Sundry observations have been recorded concerning changes in the vesicle population as a result of stimulation, The number of vesicles close to the preterminal membrane was found to diminish as a result of stimulation of the diaphra~ (Jones & Kwanbunbumpen, 1970 a) but after tetanic stimulation had depressed the quanta1 th.e electrophysiological response content, remained below normal, even though the number of vesicles close to the membrane was above control levels CiJrz;tio,&, Kwanbunbumpen, I g7o b). between vesicle size and the size of the miniature end plate potential have been reported: for example, he~cholinium-3 reduced the quanta1 size and vesicle volume (Jones & Kwanbunbumpen, rg7o a), and administration of lanthanum ions caused the appearance of cisternal and ‘ giant ‘miniature end plate potentials (Heuser, 1974). Hemicholinium-3 interferes with choline uptake and reduces acetylcholine concentration within the terminal. By the same token it would be expected to reduce the choline available for the synthesis of phospholipids required for the vesicle membrane. The rather drastic changes in membrane properties necessary to form cisternae could well cause the giant potentials without there being a causal connection between the two phenomena. Black widow spider venom (Clark, Hurlbut & Mauro, 1972) probably acts similarly, the disruption of both vesicles and terminal (see for example, Heilbronn, 1972) causing an increase in the frequency of miniature and end plate potentials in parallel with, but not as a consequence of, vesicle disappearance. These somewhat tenuous correlations between acetyIcholine release and changes in the vesicle population must be set against the evidence now available that neither the existence of the general population of vesicles
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nor those close to the membrane is a necessary condition for acetylcholine release on stimulation. Birks (1971, 1974) used I IO mM Mg2+ in the glutaraldehyde fixative and was able to show that the vesicle population of the superior cervical ganglion could be severely depleted without changes in the total amount of a~etylcholine present or the amount of acetylcholine released per stimulus. Zimmermann & Whittaker (1g74b) observed that the physiological response of the electric organ of Torpedo recovered completely after tetanic stimulation 20 hours before the terminals regained their complement of vesicles and 60 hours before the vesicular acetlycholine concentration was back to its normal value. Finally, Landmesser & Pilar (1972) noted that in the developing isolated ciliary ganglion of the chick, synaptic transmission began well before the appearance of synaptic vesicles. In summary, the morphological evidance indicates that acetylcholine release on stimulation does not occur through the agency of vesicles but that the vesicle population can be modified as a result of transmitter release. CONCLUSION How is the acetylcholine released and what is the role of the vesicles? For the reasons given in the previous section it is very dithcult to see how most of the acetylcholine could have originated directly from the synaptic vesicles that can be isolated by subcellular fractionation. There may be other types of synaptic vesicles; if so, they remain to be isolated and characterized. It would seem germane to consider other possible mechanisms such as the movement of acetylcholine down its electrochemical gradient through voltage-dependent membrane pores or carriers. There is of course no more evidence for a mechanism of this kind than there is for exocytosis. Concerning the role of vesicles, at any rate as isolated by subcellular fractionation, one may be slightly more positive. The properties of acetylcholine binding by the vesicles, with the outer, recently synthesized acetylcholine readily exchangeable and the preformed core less so, suggest that the vesicles are the
3’0
‘ reserve ’ fraction of the releasable acetylcholine described by Birks & Macintosh (I 961) and discussed in the previous section. The acetylcholine output on stimulation decreases as the readily releasable acetylcholine in the extra-vesicular compartment and on the surface of the vesicles is depleted. The acetylcholine is replenished at a slower rate from the core of the vesicle (the ‘ reserve ’ fraction of Birks & Macintosh, 1g61), and it is this rate-controlling process which sustains the steady output rate of acetylcholine after the initial sharp drop first observed by these investigators. Clearly this is a tentative model; it is incomplete and much remains to be established, but it does provide a correlation of structure and function that does not violate the salient fact.s of cholinergic transmission as they are understood at the moment. REFERENCES BABEL-GUERIN,E. ( 1g74), ‘ Metabolisme calcium et liberation de I’acetvlcholine l’organe electrique de la torpille ‘, 3. them., 23, 525-533.
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du dam Neuro-
BENNETT.M. V. L.. PAPPAS.G. D.. GIMENEZ.M. & N&AJIMA. $. (1g67), ‘ Physiology ‘and ultrastructure of electrotonic junctions IV. Medullary electromotor nuclei in Gymnotid fish ‘, 3. Neurophysiol, 30, 236-301. BIRKS, R. (rg7r), ‘ Effects of stimulation on synaptic vesicles in sympathetic ganglia as shown by fixation in the presence of Mgs+ ‘, 3. Physiol., Land., 216,26~-28~. BIRKS, R. I. (rg74), ‘ The relationship of transmitter release and storage to fine structure F33a1syompathetic ganglion ‘, 3. Neurocytol., 3,
BIRKS,R., & MACINTOSH,F. C. (1g61), ‘ Acetylcholine metabolism of a sympathetic ganglion ‘, Can. 3. Biochem. Physiol., 39, $KG-827. CANEPA, F. G. (ig64), ‘ Acetylcholine quanta ‘, Nature, Lond., 201, 184-185. CHAKRIN, L. W., MARCHBANKS, R. M., MITCHELL, J. F., & WHITTAKER, V. P. (rg72), ‘ The origin of the acetylcholine released from the surface of the cortex ‘, 3. .Neurochem., 19, 2727-2736. CLARK, A. W., HURLBUT, W. P., & MAURO, A. (rg72), ‘ Changes in the fine structure of the neuromuscular junction of the frog caused by black widow spider venom ‘, 3. Cell. Biol., 52, 1-14. COLLIER, B., & MACINTOSH, F. C. (rg6g), ‘ The source of choline for acetylcholine synthesis in a sympathetic ganglion ‘, Can. 3. Physiol. _, f%armacol., 47, 127-135.
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COLLIER, B., POON, P., & SALEHMOGHADDAM,S. ‘ The formation of choline and of (r972), acetylcholine by brain in vitro ‘, 3. Neurochem., 1g,51-60. DEL CASTILLO, J., & KATZ, B. (ig54), ‘ Quanta1 components of the end plate potential ‘, 3. Physiol., Land., 124, 560-573. DEL CASTILLO, J., & KATZ, B. (ig55), ‘ Local activity at a depolarized nerve-muscle junction ‘, 3. Physiol., Lond., 128, 396-41 I. DEL CASTILLO, .l., & KATZ, B. (1o57), _-.,. ‘ La base neuromuscu“ quantale ” -de la transmission laire ‘. in MicrobhvsioLoPie Combaree des Elements E.ucitables. Coil: i&&t. C.N.R.S. Paris. No. 67, 245-258. DE ROBERTIS. E. D. P.. & BENNETT. H. S. (>1455). __.,,, ‘ Some features of ‘the submicroscopic morphology of synapses in frog and earthworm ‘, 3. Biophys Biochem. Cytol, I, 47-58. DOWDALL. M. I.. & ZIMMERMANN, H. (1974), _. _,. ‘ Evidence fog ~heterogenous pool of acetylcholine in isolated cholinergic synaptic vesicles ‘, Brain Res., 71, 160-166. DOWDALL, M. .I., BOYNE, A. F., & WHITTAKER, ‘ Adenosine triphosphate, a (1974, constituent of cholinemic svnaotic vesicles ‘, Biochem.J., 140, 1-12. ‘ L DUNANT, T., GAUTRON, J., ISRAEL, M., LESBATS, B., & MANARANCHE, R. (rg72), ‘ Les compartiments d’acetylcholine de l’organe electrique de la torpillk et leurs modifications par la stimulation ‘,J. Neurochem., xg, Ig87-2002. DUNANT, Y., GAUTRON, J., ISRAEL, M., LESBATS, B.. & MANARANCHE, R. (19741, ~ _. _,. ’ Evolution de la- decharge de l’organec electrique de la torpille et variation simultanees de l’acetylcholine au tours de la stimulation ‘, 7. .Neurothem., 23, 635-643. ELMQUIST, D., & QUASTEL, D. M. J. (rg65), ‘ Presvnaotic action of hemicholinium at the neuro’mu&lar junction ‘, 3. Physiol., Lond., 177, 463-482. FONNUM, F. (x967), ‘ The “ compartmentation ” of choline acetyltransferase within the synaptosome ‘, Biochem. J., 103, 262-270. GRYNSZPAN-WINOGRAD, 0. (1g71), ‘ Morphological aspects of exocytosis in the adrenal medulla ‘, Phil. Trans. R. Sot. B., 261, 2g I-
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‘ Synthesis and release of HAGA, T. (rg7r), [i%]acetylcholine in synaptosomes ‘, 3. Neuro&em., 18, 781-798. HEILBRONN, E. (rg72), ‘ Action of phospholipase A on synaptic vesicles. A model for transmitter release? ’ in Progress in Brain Research (ed. Bradley & Brimblecombe) pp. 29-40. Amsterdam: Elsevier. H-&., E. (1x4, ‘ A possible origin of the spontaneous potentials that occur after prolonged transmitter release at frog neuromuscular_ junctions ‘, 3. Physiol., Lond. 239, I ob-1 o8I’.
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HEUSER, J. E., & REESE, T. S. (rg73), ‘ Evidence for recycling of synaptic vesicle membrane during transmitter .release at the frog neuromuscular junction ‘, 3. Cell Biol., 57, 3 I 5-344. HOLTZMAN, E., FREEMAN, A. R., & KASHNER, alteral L. A. (19711,‘ Stimulation-dependent tions in peroxidase uptake at lobster neuromuscular junctions ‘, Science, Jv. r., x73,733-736. HUBBARD, J. I. (rg7o), ‘ Mechanism of transmitter release ‘, Prog. Biophys. Molec. Biol., PI, 33124. ISRAEL, M., & TUBEK, S. (rg74), ‘ Utilization of acetate and pyruvate for the synthesis of ‘ total ‘, ‘ bound ’ and ‘ free ’ acetylcholine in the $;2;3organ of Torpedo ‘, 3. Nemo&em., 22, ISRAEL, M., HIRT, L., & MASTOUR-FRACHON, P. (rg73), ‘ Metabolism et echange d’acetylcholine dans les terminaisons nerveuses de l’organe electrique cle la Torpille ‘, C. R. hebd. Acad Sci., Paris., 276D, 2725-2728. JONES, S. F., & KWANBUNBUMPEN, S. (1970 a), ‘ The effects of nerve stimulation and hemicholinium on synaptic vesicles at the mammalian neuromuscular junction ‘, 3. Physiol., Lond., 22733 I-50. JONES, S. F., & KWANBUNBUMPEN, S. (1970 b), ‘ Some effects of nerve stimulation and hemicholinium on quanta1 transmitter release at the mammalian neuromuscular junction ‘, 3. Physiol., Land., 207, 5 I -6 I . KATO, A. C., KATZ, H. S., & COLLIER, B. (rg74), ‘ Absence of adenine nucleotide release from autonomic ganglion ‘, Nature, Lond., 249, 576577. KATZ, B., & MILEDI, R. (tg72), ‘ The statistical nature of the acetylcholine potential and its ;;lee;ar components ‘, 3. Physiol., Lond., 224, KATZ, B., ‘& ~MILEDI,R. (1g73), ‘ The binding of acetylcholine to receptors and its removal from the synaptic cleft ‘, 3. Physiol., Lond., 231, 54g574. KEWITZ, H., DROSS, K., & PLEUL, 0. (rg74), ‘ Choline and its metabolic successors in brain, in Central .Nervous System. (ed. Genazzani & Herken), pp. 2 1-32. ZBerlin:‘Springer. KOPIN, I. J., BREESE, G. R., KRAUSS, K. R., & WEISE, V. K. (rg68), ‘ Selective release of newly synthesized norepinephrine from the cat spleen during sympathetic nerve stimulation ‘, 3. Pharmac. ext). Ther., 161, 271-278. KRNJEVI~, K., & MITCHELL, J. F. (rg6r), ‘The release of acetylcholine in the isolated rat diaphragm ‘, 3. Physiol., Land. 155, 246-262. LANDMESSER,I;., & PILAR, G. (rg72), ‘ The onset and development of transmission in the chick ciliary ganglion ‘, 3. Physiol., Lond., 222, 6grMz&TOSn, F. C. (ig5g), ‘ Formation, storage, and release of acetylcholine at nerve endings ‘, Can. 3. Biochem. Physiol., 37, 343-356.
3”
MARCHBANKS, R. M. (rg67), ‘ Compartmentation of acetylcholine in synaptosomes ‘, B&hem. Pharmac., 16, 921-923. MARCHBANKS, R. M. (1969 a), ‘ The conversion of ‘%-choline into I%-acetylcholine in synaptosomes in vitro ‘, Biochem. Pharmac., 18, I 763-1766. MARCHBANKS, R. M. ( rg6g b), ‘ Biochemical organization of cholinergic nerve terminals in the cerebral cortex ‘, in Cellular Dynamics of the .Neuron (ed. Barondes), pp. I 15-;35. New York: Academic Press. MARCHBANKS, R. M., & ISRAEL, M. (rg71), ‘ Aspects of acetylcholine metabolism in the electric organ of Torpedo marmorata ‘, 3. Neuro-
ch.9 189439-448.
MARCHBANKS, R. M., & ISRAEL, M. (rg72), ‘ The heteogeneity of bound acetylcholine and synaptic vesicles ‘, Biochem. J., xzg, Ioag1061. MARCHBANKS, R. M., & ISRAEL, M. (rg73), ‘ The association of recently svnthesized acetvlcholine with synaptic vesicles ‘, Trans. Biochem. SOL., I, 131-134. MOLENAAR, P. C., POLAK, R. L., & NICKOLSON, ‘ Subcellular localization of V. J. (Im), newly-formed [3H]acetylcholine in rat cerebral cortex in vitro ‘, 3. .Neurochem., 21, 667-678. MILEDI, R. (1o61), ‘ From nerve to muscle ‘, Discovery, 22,442:450. MUSICK, J., & HUBBARD, J. I. (Ig72), ‘ Release of protein from mouse motor nerve terminals ‘, Nature, Lond., 237,27g-280. POTTER, L. T. (rg7o), ‘ Synthesis, storage and release of [i4C]acetylcholine in isolated rat diaphragm muscles ‘, 3. Physiol., Lond., 206, 145-166. RICHTER, -1. A., & MARCHBANKS, R. M. (1471 a), ‘ Synth&is of radioactive acetylcholine ‘from 13H1choline and its release from cerebral Lortkx slices in vitro ‘, 3. JVeurochem., 18, 6gI703. RICHTER, J. A., & MARCHBANKS, R. M. (1971 b), ‘ Isolation of [3H]acetylcholine pools by subcellular fractionation of cerebral cortex slices incubated with [3H]choline ‘, 3. YVeurochem., 18, 705-712. RITCHIE, A., & GOLDBERG, -4. M. (Ig7o), ’ Vesicular and synaptoplasmic synthesis of acetylcholine ‘, Science, YV.N.T.,169, 489-490. SILINSKY, E. M., & HUBBARD, J. I. (1973). ‘ Release of ATP from rat motor nerve terminals ‘, Nature, Lond., 243, 404-405. SMITH, A. D. (rg7t), ‘ Secretion of protein (chromogranin A and dopamine B-hydroxylase) from a sympathetic neuron ‘, Phil. Trans. R. Sot. B, 261, 363-370. ‘ On the turnover of acetylS”.“RF, B. (‘g73), choline in the brain ‘, Acta Physiol. Stand., SuPPl. 397, r-47. STRAUGHAN. D. W. (1960). ~_ ,, ‘ The release of acetylcholine from mammalian motor nerve endings ‘, Br. 3. Pharmac., 15, 417-424.
3’2
MARCHBANKS
TAUC, L., HOFFMANN, A., TSUJI, S., HINZEN, D. H. & FAILLE, L. (rg74), ‘ Transmission abolished in a cholinergic synapse after injection of acetylcholinesterase into a presynaptic neuron ‘, Nature., Land., 250, 4g6-498. WHITTAKER, V. P. (rg73), ‘ The biochemistry of synaptic transmission ‘, Naturwissenschaften, 60, 281-289. WHITTAKER, V. P., & SHERIDAN, M. N. (rg65), ‘ The morphology and acetylcholine content of isolated cerebral cortical synaptic vesicles ‘, 3. Neurochem., 12, 363-372. WHITTAKER, V. P., MICHAELSON, I. A., & KIRKLAND, R. J. A. (1g64), ‘ The separation of synaptic vesicles from nerve ending particles (‘ Synaptosomes ‘) ‘, Biochem. J., go, 293-303. WHITTAKER, V. P., ESSMAN,W. B., & DOWE, G. H. ‘ The isolation of pure cholinergic C. (r972), synaptic vesicles from the electric organs of elasmobranch fish of the family Torpedinidae ‘, Biochem. J., 128, 833-846.
Int. J.
Biochem.
WILSON, W. S., SCHULZ, R. A., & COOPER, J. R. (rg73), ‘ The isolation of cholinergic synaptic vesicles from bovine superior cervical ganglion and estimation of their acetylcholine content ‘, 3. Neurochem., 20, 659-667. ZIMMERMANN,H., & WHITTAKER, V. P. (I 974 a), ‘ Effect of electrical stimulation on the yield and composition of synaptic vesicles from the cholinergic synapses of the electric organ of Torpedo : a combined biochemical, electrophysiological and morphological study ‘, 3. Neurochem., 22,435-450. ZIMMERMANN,H., & WHITTAKER, V. P. (1974 b), ‘ Different recovery rates of the electrophysiological, biochemical and morphological parameters in the cholinergic synapses of the electric stimulation ‘, Torpedo organ after 3. JVeurochm., 22, I Iog-r I 14.
Key Word Index: Acetylcholine,
vesicles, synapses.
303
MINIREVIEW THE SU~CELLULAR
ORIGIN
RELEASED
OF THE ACETYLCHOLINE
AT SYNAPSES
R. M. MARCHBANKS Department of Biochemistry, Institute of Psychiatry, London SE5 8AF, U.K. (Received23 October 1974)
SHORTLY after the enunciation of the quanta1 theory of synaptic transmission by Del Castillo & Katz in 1954, small vesicles were observed in the synaptic region by electron microscopy (De Robertis & Bennett, 1955). It seemed reasonable, as suggested by Del Castillo & Katz (1955) that these vesicles were the morphological counterpart of the quanta and it was envisaged that they could quantitatively release their contents into the synaptic cleft by reverse micropinocytosis or exocytosis (Del Castillo & Katz, 1957). The demonstration by Whittaker, Michaelson & Kirkland ( I 964) that these vesicles did indeed contain the neurotransmitter acetylcholine completed the triad of observations, physiological, morphological and biochemical, the unification of which into a theory of transmitter storage and release is proposed by the vesicular hypothesis. Despite considerable attention from investigators for 20 years there remain difficulties with the vesicular hypothesis. In this short review an attempt wiI1 be made to assess its current status. The vesicular hypothesis has a weak and a strong form. The former merely states that the vesicles store the neurohumoral agent and must therefore release it somehow for the purpose of chemical transmission. The strong form, with which I am concerned here, states that the vesicles store a defined quantity of transmitter (the quantum) and release it quantitatively outside the terminal during the process of transmission. Although other mechanisms have been proposed, the most favoured is of acetylcholine release by fusion of the vesicle with the pre-terminal membrane and total exocytosis of the
vesicular content, This proposal has become an integral part of the strong form of the vesicular hypothesis and it is worth noting that it was inspired in a morphological tradition to explain a physiological phenomenon, but its proof or disproof must rely on biochemical arguments. It is necessary to show that the acetylcholine released on stimulation has come directly from within the vesicle without passing through the cytoplasm. PHYSIOLOGICAL EVIDENCE There have been three different approaches to determine the amount of acetylcholine in a quantum and they have given figures for the number of acetylcholine molecules ranging over three orders of magnitude. (I) Direct comparisons between the amount of acetylcholine released and the number of impulses divided by the number of end plates and the number of quanta per impulse have been reported by several authors (McIntosh, 1959; Straughan, 1960, KrnjeviC & Mitchell, 196x). The values range between 498 and ~oo,ooo molecules of acetylcholine per quanta. Some of the discrepancies are considered by Hubbard (1970) and a preferred figure of 40,000 emerges. (2) A comparison can be made of the content of releasable acetylcholine in the nerve terminal and the number of quanta released by stimulation to exhaustion when synthesis of acetylcholine is blocked. This procedure yields a value of 57,000 molecules of acetylcholine per quanta (Elmquist & Quastel, r 965). (3) Attempts to mimic the miniature end
304
MARCHBANKS
plate potential by electrophoretic application of acetylcholine suggest that 100,000 molecules of acetylcholine are needed (Miledi, 196 I) but presumably this is an upper limit because the injecting micropipette cannot be placed as advantageously as the site of release of acetylcholine. Somewhat different results have been obtained by the analysis of acetylcholineinduced membrane noise (Katz & Miledi, 1972). It is assumed that the noise arises from random variations in the rate of molecular collision between molecules of acetylcholine and receptor on the postsynaptic membrane. Each successful collision produces a voltage change which contributes to the amplitude of the acetylcholineinduced membrane noise measured as the variance of the membrane potential. Making certain corrections for non-linear summation, Katz & Miledi (1972) were able to calculate the characteristics of the elementary acetylcholine current pulse. Each collision of a single acetylcholine molecule with the receptor produced a conductance change of 10-10 mhos lasting for 10-3 seconds, which caused a depolarization of 0.3 PV and was about 1-2000 times smaller than the miniature end plate potential. In a further study, Katz & Miledi (1973) estimate that about 60% of the molecules of acetylcholine that are released into the synaptic cleft are bound to receptor. They conclude that about 30,000 molecules (i.e. 60% of the 50,000 molecules in the quantum estimated as described previously) are bound to receptor but that only 5-1oO/~ of the acetylcholine-receptor complex is in the active form that allows the passage of ions across the membrane. This supposes a very ‘ inefficient ’ acetylcholine receptor and an alternativeexplanationisthattheacetylcholine receptor complex approaches a reasonable efficiency but that the figure of 50,000 molecules of acetylcholine per quantum is a gross overestimate. This could be because a large amount of acetylcholine is released on stimulation in a non-quanta1 fashion but is attributed to quanta in the calculation, In fact thus causing the overestimate. 50,000 molecules can only be packed inside
Int. J. Biochem.
a vesicle if the acetylcholine is crystalline (Canepa, I 964) and the biochemical evidence (see below) suggests that mammalian synaptic vesicles contain much less acetylcholine than this. These uncertainties make it difficult to try and define a mechanism of release the physiological which would satisfy observations. BIOCHEMICAL EVIDENCE The isolation of synaptic vesicles and the demonstration that they contain acetylcholine (Whittaker et al., 1964) is probably the single most important result of biochemical studies on the topic. Without this demonstration the role ascribed to the vesicles in chemical transmission would be largely hypothetical. Vesicles from the cholinergic synapses have been isolated from three tissues; cerebral cortex of laboratory rodents, electric organ of Torpedo marmorata and from bovine superior cervical ganglion. In each case the acetylcholine content of the individual vesicles has been estimated by adding a known amount of polystyrene latex beads to the preparation so that the ratio of beads to vesicles in the field of an electron micrograph can be used to calculate the number of vesicles in the preparation and hence the ratio of acetylcholine molecules to vesicles. Synaptic vesicles from mammalian tissues contain between IOOO and 2000 molecules of acetylcholine (Whittaker & Sheridan, 1965; Wilson, Schulz & Cooper, 1973) while those from Torpedo electric organ contain rather more (Whittaker, Essman & Dowe, ‘972). The vesicles lose their acetylcholine if submitted to hypo-osmotic conditions and this is most conveniently demonstrated by gel-filtration in solutions of different osmolarity (Marchbanks, 1967; Marchbanks & Israel, rg7 I). One may conclude from this that the acetylcholine is sequestered by a semi-permeable membrane and is therefore within the vesicle rather than merely absorbed to the surface. If the concentration of acetylcholine inside the vesicle is calculated it is found to be rather high, ranging about iso-tonicity. Information about how the acetylcholine
197536
ACETYLCHOLINE
is taken up and retained within the vesicle is incomplete. A study of the association of recently synthesized acetylcholine with synaptic vesicles has offered some clues to the nature of the process. When radioactive acetylcholine is synthesized from labelled precursor in a closed system the more recently synthesized stores will have a higher specific radioactivity. It was noticed (Marchbanks & Israel, 197 r) that the acetylcholine of vesicles isolated from tissue incubated with labelled precursor displayed heterogeneity on gel-filtration. The recently synthesized acetylcholine of high specific radioactivity was lost preferentially from the vesicles when they were passed over iso-osmotically eluted Sephadex columns, whereas the non-radioactive preformed acetylcholine was retained. Using the technique of gel filtration in this way it was possible to distinguish the loosely bound recently synthesized store from the tightly bound preformed store of acetylcholine and to compare their properties (Marchbanks & Israel, 1973). It was found that the loosely bound acetylcholine could become tightly bound under various conditions but the extent of the conversion to the tightly bound form was always positively correlated with losses from the original tightly bound form. For example, incubating at pH 5 or with calcium ions increased the incorporation of radioactive acetylcholine from the loosely bound store, but at the same time losses from the tightly bound store were increased. Incubation at 6” C or 20’ C produced comparatively little change in either store. Furthermore, factors at present unknown that positively affected exchange from the loosely bound store into the tightly bound store positively affected exchange out of the tightly bound store with high statistical significance (Marchbanks & Israel, I 972). The most parsimonious explanation is that the exchanges in each direction are coupled. The coupling of the exchange between recently synthesized loosely bound acetylcholine and the preformed store of tightly bound vesicular acetylcholine must imply that both stores exist in the same vesicle, because free acetylcholine could not diffuse
RELEASE
305
between vesicles without being hydrolyzed by residual cholinesterase present in the preparation. The results are best explained by postulating that the recently synthesized loosely bound acetylcholine exists on or near the surface of the vesicle and that it cannot exchange into the tightly bound form without the vacation of binding sites in the core of the vesicle by the pre-formed and hitherto tightly bound acetylcholine. No association of the loosely bound acetylcholine with membrane fragments could be discerned. A study of the relationship between ATP (Marchbanks & Israel, 1972) and acetylcholine of vesicles (Dowdall & Zimmermann, 1974) also suggests two vesicular pools of the transmitter, one identified as being labile, rapidly lost when the electric organ is stimulated, having little associated ATP and being possibly similar to the loosely bound compartment, while the other is more stable and has an acetylcholine: ATP molar ratio of about 4 : I. Quantitatively the rate of exchange between compartments is small, so that pre-labelled vesicles are required to demonstrate it. It is probable that the action of calcium ions in hastening the rate of exchange and hence release of acetylcholine from the vesicles is of importance in the mechanisms of synaptic transmission. Stimulation of the electric organ results in entry of calcium into the pre-synaptic tissues and the progressive increase of calcium concentration is accompanied by a progressive decrease in the bound (vesicular) acetylcholine (Babel-Guerin, I 974). It seems likely that there is no hard and fast distinction between the loosely bound and tightly bound store: the acetylcholine of each concentric shell progressing inwards towards the core of the vesicle is progressively more tightly bound. Conversely progressing outwards, the acetylcholine of each concentric shell is more and more labile and also the rate at which the equilibrium between it and the extra-vesicular acetylcholine is achieved becomes progressively more rapid. In order to explain the phenomena of the exchanges, the ability of vesicles to store acetylcholine and their osmotic sensitivity, the vesicle membrane would have to be more
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MARCHBANKS
permeable to acetylcholine than to its counter-ions within the vesicle. Subcellular fractionation studies show that choline acetyltransferase is a soluble enzyme unattached to synaptic vesicles or other organelles. (Fonnum, I 967 ; Marchbanks & Israel, 1972). The site of synthesis of the transmitter is therefore in the cytoplasm. The relative rates of synthesis compared with storage in vesicles or disposal will define the size of the extra-vesicular pool ofacetylcholine. This pool comprises some 20-50% of the total, and is not derived from the breakdown of vesicles and release of their acetylcholine because in labelling experiments it always has a different specific radioactivity. Nor is it described by the ‘ surplus ’ acetylcholine Birks & Macintosh (196 I) because its presence can be demonstrated without the aid of anti-cholinesterases. The localization of the recently synthesized acetylcholinehasbeeninvestigated bystudying the subcellular disposition of radioactive acetylcholine after the tissue has been allowed to synthesize transmitter from labelled precursors. The results on isolated tissues (Marchbanks, rg6g a; Ritchie & Goldberg, 1970; Haga, 197 I ; Richter & Marchbanks, 1971, b; Marchbanks & Israel, 1971, 1972; Collier, Poon & Salechmoghaddam, 1972 ; Molenaar, Polak & Nickolson, 1973 ; Israel, Hirt & Mastour-Frachon, 1973; Israel & Tucek, 1974) can be summarized thus: the rate of incorporation into the vesicular compartment is usually less than, sometimes equal to but never greater than the rate into Experithe extra-vesicular compartment. ments of this kind with the tissue in situ have only been done on brain. Their interpretation is complicated by the permeability barriers presented to the entry of radiolabelled choline and the difficulty of identifying the period of peak incorporation after the pulse of labelled precursor. The turnover rate is so fast that the initial peak cannot easily be observed and the simplest explanation of the results of Sparf (1973) and Kewitz, Dross & Pleul (1974) would seem to be that the label in the extra-vesicular compartment of acetylcholine is diluted out faster with recently synthesized non-radioactive acetylcholine
ht.
3.
Biochem.
than is the label in the vesicular compartment, indicating acetylcholine synthesis outside the vesicles. These results suggest that, as would be expected from the localization of choline the most recently synacetyltransferase, thesized acetylcholine is extra-vesicular but that it can be transferred to the vesicular compartment quite rapidly under certain It is unlikely that empty circumstances. vesicles are filled with newly synthesized acetylcholine providing a small population of highly labelled vesicules whose radioactivity is swamped by a larger population of vesicles containing preformed unlabelled acetylcholine, because as explained previously, the newly synthesized acetylcholine, insofar as it exists in the vesicle fraction, appears to be on the surface of each vesicle. There is no discrete population of highly labelled vesicles that with present techniques can be isolated by subcellular fractionation. The studies of Birks & Macintosh (1961) on the superior cervical ganglion identified two compartments of acetylcholine available for release by stimulation. The ‘ readily contains about releasable ’ compartment 20% of the total releasable acetylcholine and this can be released on stimulation; the so-called ‘ reserve pool ’ contains about 80% of the total releasable acetylcholine, which is fed into the ‘ readily releasable ’ compartment, making acetylcholine available for release at a steady rate. The relationship between the readily releasable acetylcholine and the recently synthesized acetylcholine was first established by Collier & Macintosh (1969). The acetylcholine of the superior cervical ganglion was completely labelled by perfusion with [3H]choline-Locke solution; perfusion was then continued with unlabelled choline and the ganglion stimulated. The acetylcholine that was released had a lower specific activity than that remaining in the ganglion stores. It was concluded that the acetylcholine recently synthesized from the unlabelled choline was preferentially released by the stimulation. This implies that the reserve store exists in a cul-de-sac from the main path of acetylcholine throughput. This important result had also been
1975>6
ACETYLCHOLINE
suggested by studies on the evoked release of [3H]acetylcholine from the surface of the cerebral cortex (Marchbanks, 1969 b; Chakrin, Marchbanks, Mitchell & Whittaker, 1972). In addition, studies on the acetylcholine released from cerebral cortex slices by high potassium concentrations also suggested that it came from a newly synthesized compartment (Richter & Marchbanks, 1971 a; Molenaar et al., Ig73), although in interpretating this result one should not assume that potassium and electrical stimulation are exactly equivalent. The preferential release of newly synthesized acetylcholine on stimulation takes place in the diaphragm (Potter, 1970) and also in the electric organ of Torpedo (Dunant, Gautron, Israel, Lesbats & Manaranche, 1972). Splenic nerve stimulation releases newly synthesized noradrenaline from the spleen (Kopin, Breese, Krauss & Weise, 1968) and this suggests that the phenomenon, in addition to being a well established feature of cholinergic systems, is also a general characteristic: of neurohumoral transmission. It will be recalled from the previous section that the extra-vesicular acetylcholine is more recently synthesized than that within the vesicles. Therefore it follows that the vesicle population as isolated does not represent the immediate source of releasable acetylcholine, since to be released by exocytosis the recently synthesized acetylcholine would have to be present in the core of the vesicle, whereas it actually exists free and on the surface of the vesicle. Much more direct and striking evidence concerning the origin of the released acetylcholine comes from the studies of Dunant and his colleagues at the Salp&tri&-e Hospital (Dunant et al., These ‘972). investigations stimulated the electric organ of Torpedo either in situ or when cut in slices and superfused with physiological salines. They then immediately determined the ‘ free ’ and ‘ bound ’ acetylcholine that remained resistant to hydrolysis when the tissue was homogenized. The bound acetylcholine had previously been shown to be equivalent to vesicular acetylcholine in Torpedo, because there is no formation of
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307
synaptosomes and the specific radioactivity of acetylcholine in the bound and vesicular compartments were found to be the same (Marchbanks & Israel, 197 I). The immediate result of stimulation was a drop in the concentration of free acetylcholine; prolonged periods of stimulation eventually caused a drop in the bound acetylcholine. The electrophysiological response to stimulation was initially a 3oV depolarization but it decreased rather rapidly to about 50% within one hundred stimuli. Removal of calcium from the medium or addition of a high magnesium concentration prevented the drop in the concentration of free acetylcholine and also reduced the electrophysiological manifestation (see also Babel-Guerin, ‘974). In further studies (Dunant, Gautron, Israel, Lesbats & Manaranche, 1974) it was shown that the decrease in the electrophysiological response was exactly paralleled by the decrease in free acetylcholine over the course of the first IOO stimuli; subsequently the free acetylcholine concentration increased and this was associated with a plateau in the decrease of the electrophysiological response. The bound acetylcholine concentration did not change during these events, and experiments with [‘*C]choline showed that there was no increased exchange of acetylcholine, so that rapid emptying and refilling of the vesicles with acetylcholine can be ruled out. Further prolonged stimulation did then cause a decrease in the bound acetylcholine and changes in the number of vesicles observable in the synaptic region. The results of Zimmermann & Whittaker (1974 a) confirm those of Dunant et al. (1972) in showing that after prolonged stimulation ( > 500 stimuli) there was a loss of vesicular acetylcholine. However they did not measure extra-vesicular acetylcholine and they proposed that acetylcholine has been released by exocytosis. In their next paper (Zimmermann & Whittaker, 1974 b) findings are reported that are inconsistent with this suggestion (see below). In the adrenergic system, evidence for exocytosis is easier to obtain because the vesicles contain soluble protein such as
303
Int. J. Biochem.
MARCHBANKS
dopamine-fl-hydroxylase and chromogranin A. On stimulation of the splenic nerve, protein appears in the perfusate along with noradrenaline and this is taken to indicate exocytosis, because it is difficult to see how a protein could be released by any other process (Smith, 1971). It is noteworthy that much more noradrenaline relative to dopamine-,%hydroxylase is found in the perfusate than in the vesicles, suggesting that exocytosis is not the only release process. Analogous evidence for exocytosis in the cholinergic system is more difficult to obtain, because, despite some claims (cf. Whittaker, it has not been established that ‘973L cholinergic vesicles specifically contain a unique soluble protein. The observation by Musick & Hubbard (1972) that proteins (unidentified and of unknown provenance) are released from the hemidiaphragm on stimulation is therefore difficult to interpret in this context. However cholinergic vesicles appear to contain ATP (Dowdall, Boyne & Whittaker, 1974) and the Ca2+-dependent release of ATP on stimulation of the diaphragm (Silinsky & Hubbard, 1973) is therefore of interest. As evidence for exocytosis, the release of ATP is less persuasive than the release of a specific vesicular protein, because the concentration in the cytoplasm is at least IO times higher (Dowdall et al., I 974) than in the vesicles. ATP release could not be detected when the superior cervical ganglion was stimulated (Kato, Katz & Collier, 1974). The biochemical evidence concerning the cholinergic synapse therefore not only does not support exocytosis but also suggests that the extra-vesicular acetylcholine is first and preferentially lost on stimulation. The theory of exocytosis could be preserved if there were a population of labile vesicles which were preferentially filled with acetylcholine and which preferentially released it on stimulation and which also were not stable enough to survive tissue homogenization. As already remarked there is no biochemical evidence for this proposal. The accuracy of the measurements do not warrant the statement that exocytosis never occurs but it does not contribute the majority of the acetylcholine
released on stimulation. A recent experiment by Taut, Hoffmann, Tsuji, Hinzen & Faille (1974) demonstrated that transmission could be abolished by the injection into the cell of Acetylcholine inside acetylcholinesterase. the vesicles is protected from hydrolysis by the enzyme so it follows that the extravesicular acetylcholine is responsible for the physiological response in this experiment. MORPHOLOGICAL
EVIDENCE
Despite the tradition within which the vesicular hypothesis was developed, the morphological evidence has always been rather scanty and contradictory. Until recently the event of exocytosis had only been rarely visualized and even then only in systems (unlike the cholinergic system) where the core of the vesicle is electron-dense (GrynszpanWinograd, 1971). What has been demonstrated with the aid of an electron-dense reaction product of horseradish peroxidase is pinocytosis at synapses (Holtzman, Freeman & Kashner, 1971; Heuser & Reese, 1973). The latter authors observe that on stimulation, reaction product is seen in coated vesicles formed at the edge of the synapse, presumably by pinocytosis. The coated vesicles fuse to form cisternae and after a period the reaction product is found in synaptic vesicles from whence it disappears during further stimulation. In addition, Heuser & Reese (1973) show that on stimulation, synaptic vesicles disappear and the terminal membrane gains (supposedly by exocytotic fusion of the vesicle) an equivalent amount of membrane which is subsequently transferred to the cisternae. However, Birks (1974) was not able to observe any expansion of the terminal area cervical ganglion on in the superior stimulation. The weakest link in this elegantly observed chain is that relating to the loss of horseradish peroxidase from the vesicles. It could have disappeared by mechanisms other than exocytosis, perhaps enzymic degradation, for example. Furthermore, even if the enzyme were expelled by exocytosis there is no reason to suppose that these particular vesicles contain acetylcholine or that the process is representative of those
197.s6
ACETYLCHOLINE
that do. It is worth recalling in this connection the synapses that are thought to be electrically transmitting and therefore presumably do not contain stores of transmitter are nevertheless well supplied with synaptic vesicles (Bennett, Pappas, Gimenez & Nakajima, rg67), Sundry observations have been recorded concerning changes in the vesicle population as a result of stimulation, The number of vesicles close to the preterminal membrane was found to diminish as a result of stimulation of the diaphra~ (Jones & Kwanbunbumpen, 1970 a) but after tetanic stimulation had depressed the quanta1 th.e electrophysiological response content, remained below normal, even though the number of vesicles close to the membrane was above control levels CiJrz;tio,&, Kwanbunbumpen, I g7o b). between vesicle size and the size of the miniature end plate potential have been reported: for example, he~cholinium-3 reduced the quanta1 size and vesicle volume (Jones & Kwanbunbumpen, rg7o a), and administration of lanthanum ions caused the appearance of cisternal and ‘ giant ‘miniature end plate potentials (Heuser, 1974). Hemicholinium-3 interferes with choline uptake and reduces acetylcholine concentration within the terminal. By the same token it would be expected to reduce the choline available for the synthesis of phospholipids required for the vesicle membrane. The rather drastic changes in membrane properties necessary to form cisternae could well cause the giant potentials without there being a causal connection between the two phenomena. Black widow spider venom (Clark, Hurlbut & Mauro, 1972) probably acts similarly, the disruption of both vesicles and terminal (see for example, Heilbronn, 1972) causing an increase in the frequency of miniature and end plate potentials in parallel with, but not as a consequence of, vesicle disappearance. These somewhat tenuous correlations between acetyIcholine release and changes in the vesicle population must be set against the evidence now available that neither the existence of the general population of vesicles
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RELEASE
nor those close to the membrane is a necessary condition for acetylcholine release on stimulation. Birks (1971, 1974) used I IO mM Mg2+ in the glutaraldehyde fixative and was able to show that the vesicle population of the superior cervical ganglion could be severely depleted without changes in the total amount of a~etylcholine present or the amount of acetylcholine released per stimulus. Zimmermann & Whittaker (1g74b) observed that the physiological response of the electric organ of Torpedo recovered completely after tetanic stimulation 20 hours before the terminals regained their complement of vesicles and 60 hours before the vesicular acetlycholine concentration was back to its normal value. Finally, Landmesser & Pilar (1972) noted that in the developing isolated ciliary ganglion of the chick, synaptic transmission began well before the appearance of synaptic vesicles. In summary, the morphological evidance indicates that acetylcholine release on stimulation does not occur through the agency of vesicles but that the vesicle population can be modified as a result of transmitter release. CONCLUSION How is the acetylcholine released and what is the role of the vesicles? For the reasons given in the previous section it is very dithcult to see how most of the acetylcholine could have originated directly from the synaptic vesicles that can be isolated by subcellular fractionation. There may be other types of synaptic vesicles; if so, they remain to be isolated and characterized. It would seem germane to consider other possible mechanisms such as the movement of acetylcholine down its electrochemical gradient through voltage-dependent membrane pores or carriers. There is of course no more evidence for a mechanism of this kind than there is for exocytosis. Concerning the role of vesicles, at any rate as isolated by subcellular fractionation, one may be slightly more positive. The properties of acetylcholine binding by the vesicles, with the outer, recently synthesized acetylcholine readily exchangeable and the preformed core less so, suggest that the vesicles are the
3’0
‘ reserve ’ fraction of the releasable acetylcholine described by Birks & Macintosh (I 961) and discussed in the previous section. The acetylcholine output on stimulation decreases as the readily releasable acetylcholine in the extra-vesicular compartment and on the surface of the vesicles is depleted. The acetylcholine is replenished at a slower rate from the core of the vesicle (the ‘ reserve ’ fraction of Birks & Macintosh, 1g61), and it is this rate-controlling process which sustains the steady output rate of acetylcholine after the initial sharp drop first observed by these investigators. Clearly this is a tentative model; it is incomplete and much remains to be established, but it does provide a correlation of structure and function that does not violate the salient fact.s of cholinergic transmission as they are understood at the moment. REFERENCES BABEL-GUERIN,E. ( 1g74), ‘ Metabolisme calcium et liberation de I’acetvlcholine l’organe electrique de la torpille ‘, 3. them., 23, 525-533.
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Key Word Index: Acetylcholine,
vesicles, synapses.