before reviewing the results obtained with these preparations , those with brain synaptic vesicles should be briefly mentioned . nerve terminals take up about 3% of cortical brain mass , so the discovery ( gray and whittaker 1960 , 1962 ) that they could be isolated as sealed structures to which we later gave the name synaptosomes provided us , inter alia , with a preparation enriched over 30-fold with synaptic vesicles . these we could isolate from them in a high state of purity ( whittaker and sheridan 1965 ) . by labeling our preparations with known amounts of vesicle - sized polystyrene beads , we were able to determine the average number of ach molecules per vesicle as 300 . since cholinergic endings comprise only about 3% of the total , the molecular ach content of cholinergic vesicles must be about 6,000 , close to the best estimates of the size of the quantum ; this figure is also consistent with a synaptic vesicle filled with an iso - osmotic ach solution . using a simplified version of a purification technique developed in my lab ( nagy et al . 1976 ) and using techniques not available to us , jahn and coworkers ( takamori et al . 2006 ) have been able to construct what amounts to a molecular model of the synaptic vesicle . however , this is a model , not of the cholinergic vesicle , but of the predominant kinds , those from glutamergic and gabaergic terminals , which , as we have seen , greatly outnumber cholinergic ones . it contains seven known proteins or groups of protein subunits : the vesicular proteoglycan ( vpg ) whose negatively charged glycan moieties fill the vesicle core ; the ach and adenosine triphosphate ( atp ) transporters ( ach - t , atp - t ) ; the vesicular ( v - type ) proton - translocating adenosine triphosphatase ( v - atpase ) , which provides , via proton exchange , the driving force for ach uptake , and indirectly , for that of atp ; the p - type atpase ( p - atpase ) responsible for regulating the ca and mg content of the vesicle ; synaptophysin , possibly an ion channel ; and -actin , possibly a link to the cytoskeleton , permitting vesicle movement . in addition , very likely though not positively identified , there is an eighth protein  synaptobrevin  which is present in brain vesicles and acts as a hook , attaching the vesicle to the cytoplasmic surface of the plasma membrane , a necessary preliminary to vesicle  docking and exocytosis . figure  1composition of the reserve ( v1 ) synaptic vesicle from the cholinergic electromotor synapse . number of copies of membrane constituents , if above three and where known , are given in parentheses . for abbreviations , see text composition of the reserve ( v1 ) synaptic vesicle from the cholinergic electromotor synapse . number of copies of membrane constituents , if above three and where known , are given in parentheses . for abbreviations , see text vpg can be detected immunohistochemically or by s labeling of its sulfate groups . using these markers we have shown that the vesicles are formed in the cell body , travel down the axon by fast axonal transport , and form a pool of newly arrived vesicles of low ach content ( fig .  2 ) . the glycan moieties of vpg may act like an ion - exchange resin ; during exocytosis ach is exchanged for inorganic ions from the extracellular fluid . recovered vesicles rapidly exchange ach for na and k ions and , if stimulation continues , undergo repeated exo- and endocytosis . these recycling vesicles constitute the v2 pool . during an ensuing period of rest , bivalent ions that have blocked the negative charges of vpg ate exchanged via the p - atpase for more ach ; the resultant increase in the free solute load causes water to enter and the vesicles to swell and become less dense . these changes in ionic composition and accompanying changes in water content cause differences in the sizes and densities of the three vesicle pools which make their physical separation possible ( table  1 ) . figure  2cartoon representing the three pools of synaptic vesicles in the cholinergic nerve terminal , their maturation , mobilization , recycling , recovery , and eventual demobilization . for explanation and abbreviations , see texttable  1electromotor synaptic vesicles are heterogeneous in size , density , and functionpoolv0v1v2functional stateaxonalreserverecyclingach molecules per vesicle200246,00079,000inorganic ionshighlowhighfree water ( % v1)656542diameter ( nm)909070density ( g / ml)1.0501.0561.062atpase ( nmol / h/100  u synaptophysin)240690n / dwhittaker ( 1990 ) cartoon representing the three pools of synaptic vesicles in the cholinergic nerve terminal , their maturation , mobilization , recycling , recovery , and eventual demobilization . for explanation and abbreviations , see text electromotor synaptic vesicles are heterogeneous in size , density , and function after an unknown number of cycles of exo- and endocytosis , vesicles become demobilized and their membranes are salvaged via the endosomal system for reprocessing . in electromotor terminals , coated vesicles are rarely seen , but endosome - like membranes and depleted vesicle numbers are seen after prolonged stimulation . the lipid and protein compositions of the plasma and vesicle membranes are known to remain distinct ( whittaker 1966 ) ; this shows that the retrieval of immobilized vesicle membrane is efficient and conservative . the trigger for demobilization is unknown but it may be the loss of glycan residues from vpg . released ach , after combining with its receptors , is rapidly hydrolysed to acetate and choline and salvaged by the presynaptic terminal . choline is reacetylated by the cytoplasmic enzyme choline acetyltransferase ( chat ) , providing a pool of cytoplasmic ach . 1982 ) . marked differences in the specificities of chat and vesicular ach - t allow the cytoplasmic and vesicular pools to be differentially labeled with choline analogs and compared with the labeled transmitter released on stimulation . such experiments show that , in the resting synapse , there is little exchange between the cytoplasmic and v1 pools , and that the source of released transmitter is the recycling vesicular and not the cytoplasmic or reserve vesicular pools ( whittaker and luqmani 1980 ; luqmani et al . work with mplm ( agoston et al . 1985 ) has shown that in this tissue too v1 and v2 vesicle pools exist but , due to the small size of the vesicles , they differ less in density and so are less well separated by density gradient centrifuging that the corresponding two pools of electromotor vesicles . we prepared presynaptic plasma membranes from torpedo electric organs and injected them into sheep . the resulting anti - serum , after purification by adsorption , proved to be specific for cholinergic neurones in the mammalian brain . figure  3 shows one of a number of ways in which this was demonstrated ( data plotted from richardson 1983 ) . synaptosomes , like cells , can be sensitized to lysis by complement by pretreating them with an anti - serum which recognizes an antigen on the plasma membrane . a mammalian brain cortical synaptosome preparation was treated with our putative cholinergic - specific anti - serum , complement applied and the release of various transmitter - specific cytoplasmic markers measured . for cholinergic synaptosomes the marker was chat ; glutamergic , gabaergic , serotonergic , noradrenergic , and dopaminergic synaptosomes were loaded with the appropriate radio - labeled cytoplasmic transmitter precursor . radio - labeled deoxyglucose ( dog ) served as a non - specific cytoplasmic marker and an anti - cortex anti - serum as a non - specific sensitizer . figure  3the selective complement lysis of the cholinergic subpopulation of brain cortical synaptosomes after sensitization by an anti - chol-1 anti - serum . radio - labeled transmitter - specific cytoplasmic markers : glu glutamic , gaba -aminobutyric acids , 5-ht serotonin , na noradrenaline , da dopamine . for other abbreviations , see text the selective complement lysis of the cholinergic subpopulation of brain cortical synaptosomes after sensitization by an anti - chol-1 anti - serum . radio - labeled transmitter - specific cytoplasmic markers : glu glutamic , gaba -aminobutyric acids , 5-ht serotonin , na noradrenaline , da dopamine . for other abbreviations , see text it will be seen that of all the transmitter - specific cytoplasmic markers , only chat is released when the preparation is sensitized by the putative cholinergic - specific anti - serum . significantly , this release is accompanied by the release of about 5% of dog , about the proportion of cholinergic synaptosomes present in the preparation . by contrast , all the synaptosomes are sensitized by the anti - cortex anti - serum as shown by the extensive release of dog in this case . work by japanese colleagues ( for refs see whittaker and kelic 1995 ) has identified the antigen recognized by our anti - serum , which we called chol-1 , as a hitherto unknown family of brain gangliosides , characterized by sialylation of the n - acetylgalactosamine residue ( table  2 ) . although these gangliosides account for only 0.1% of total brain gangliosides , when allowance is made for the small contribution that cholinergic endings make to total brain mass , the chol-1 gangliosides could be a major constituent of the gangliosides of the cholinergic presynaptic terminal membrane . the anti - chol-1 anti - serum has been applied to the isolation of the cholinergic subfraction of brain cortical synaptosomes ( agoston et al . , the synaptosomes were passed through a column of an immobilized high - titre monoclonal mouse anti - sheep igg antibody . the non - cholinergic synaptosomes had not bound anti - serum , so were not recognized by the column and could be washed through , leaving the cholinergic synaptosomes attached to the column . the enrichment of ach and chat corresponded to an expected ca 5% abundance of cholinergic synaptosomes in the whole population ( table  3 ) . by contrast , lactate dehydrogenase ( ldh ) , a general cytoplasmic marker , was not enriched . table  3copurification of vip with cholinergic rat brain cortical synaptosomes affinity - purified using sheep anti - chol-1 anti - serum and immobilized rat anti - sheep iggcomponentpurification factoryield ( % ) ach19chat153.3vip84.0ldh1agoston et al . ( 1988a ) copurification of vip with cholinergic rat brain cortical synaptosomes affinity - purified using sheep anti - chol-1 anti - serum and immobilized rat anti - sheep igg agoston et al . ( 1988a ) interestingly , the neuropeptide vasoactive intestinal polypeptide ( vip ) is also enriched , but only to half the extent of the cholinergic marker . long suspected from immunocytochemical observations to be associated with cholinergic neurones , these results suggest that 50% of cholinergic synaptosomes contain vip . however , the yield of vip is higher than that of chat , suggesting that 70% of vip - containing endings also contain chat , i.e. , are also cholinergic . vip is also found in association with cholinergic neurones in the peripheral nervous system , the electromotor system being no exception ( agoston et al . throw away vesicles which , unlike synaptic vesicles , can not be refilled in the periphery and are thus entirely dependent on axonal transport for replenishment . this is well illustrated by some work we did with the mplm preparation ( agoston et al . the release of vip on stimulation was quickly blocked by colchicine , an axonal transport blocker , while that of acetylcholine was unaffected over a similar period . by contrast , hemicholinium , which blocks the uptake of choline by nerve terminals and so inhibits the synthesis of ach and its uptake by recycling vesicles , quickly brought its release to an end while leaving that of vip unaffected . table  4 shows that vip , while it is the peptide most often associated with cholinergic neurones , is by no means the only ones to be so . the difference in the dynamics of release of neuropeptides and small molecular mass transmitters may suggest that the neuropeptides are a kind of . 
 table  4co - localization of neuropeptides in cholinergic neuronsfunction / localizationspeciesneuropeptidemotor / secretomotor  parasympathetic gangliacatvip  myenteric plexus- longitudinal muscleguinea pigvip , sp , somatostatincholecystokininneuropeptide y  hypoglossal & facial nuclei , guinea pigcgrp  nucleus ambiguusvasomotor  cerebral cortexratvipelectromotor  electromotor neuronstorpedo sppvip , grpsensory  superior oliveguinea pigenkephalinactivation system  lateral dorsal segmentalratsphigher brain function  medial septal neurons , diagonal band , ratgalantine  hippocampal afferentsfor references see whittaker ( 1992)cgrp calcitonin - gene - related peptide , grp gastrin - releasing peptide , sp substance p , vip vasoactive intestinal polypeptide co - localization of neuropeptides in cholinergic neurons for references see whittaker ( 1992 ) cgrp calcitonin - gene - related peptide , grp gastrin - releasing peptide , sp substance p , vip vasoactive intestinal polypeptide those interested in exploring the topics raised in this article may consult whittaker ( 1992 ) and whittaker and kelic ( 1995 ) for references and further results .
at the request of the organizers of the xiii international symposium on cholinergic mechanisms , i have selected for review three topics not well represented at the meeting : the synthesis , storage , and release of acetylcholine at cholinergic nerve endings ; cholinergic - specific antigens ; and neuropeptides as cholinergic co - transmitters . the first topic is illustrated by work with two model systems , the electromotor nerve terminals of the electric ray and the myenteric plexus of the guinea - pig ileum , the second by work with chol-1 , a group of cholinergic - specific gangliosides sialylated on their n - acetylgalactosamine residues , and the third by vasoactive intestinal polypeptide which is a co - transmitter at 70% of cholinergic nerve endings in brain and is also present in both of the model cholinergic systems .