DEPLETION OF VESICLES FROM FROG NEUROMUSCULAR JUNCTIONS BY PROLONGED TETANIC STIMULATION

Curarized cutaneous pectoris nerve muscle preparations from frogs were subjected to prolonged indirect stimulation at 2/sec while recording from end plate regions. At the ends of the periods of stimulation, the curare was removed and the preparations were fixed for electron microscopy or treated with black widow spider venom to determine the degree to which their stores of transmitter had been depleted. After 6–8 hr of stimulation the nerve terminals were almost completely depleted of their stores of transmitter and of their population of vesicles. Most of the transmitter release occurred during the first 4 hr of stimulation, and after this time most (about 80%) of the fibers were depleted of about 80% of their transmitter. The organization of the nerve terminals in 4-hr preparations appeared normal and the terminals still contained many vesicles. When peroxidase was present in the bathing medium, terminals from stimulated preparations showed many vesicles that contained peroxidase, whereas the rested control preparations showed few such vesicles The fact that after 4 hr the total number of vesicles is not markedly changed while a large fraction (up to 45%) contained peroxidase suggests that in our experiments vesicles were continuously fusing with and reforming from the axolemma.     

Mobilization of the Readily Releasable Pool of Acetylcholine from a Sympathetic Ganglion by Tityustoxin in the Presence of Vesamicol

The present experiments tested whether preganglionic stimulation and direct depolarization of nerve terminals by tityustoxin could mobilize similar or different pools of acetylcholine (ACh) from the cat superior cervical ganglia in the presence of 2-(4-phenylpiperidino)cyclohexanol (vesamicol, AH5183), an inhibitor of ACh uptake into synaptic vesicles. In the absence of vesamicol, both nerve stimulation and tityustoxin increased ACh release. In the presence of vesamicol, the release of ACh induced by tityustoxin was inhibited, and just 16% of the initial tissue content could be released, a result similar to that obtained with electrical stimulation under the same condition. When the impulse-releasable pool of ACh had been depleted, tityustoxin still could release transmitter, amounting to some 10% of the ganglion's initial content. This pool of transmitter seemed to be preformed in the synaptic vesicles, rather than synthesized in response to stimuli, as tityustoxin could not release newly synthesized [3H]ACh formed in the presence of vesamicol, and hemicholinium-3 did not prevent the toxin-induced release. In contrast to the results with tityustoxin, preganglionic stimulation could not release transmitter when impulse-releasable or toxin-releasable compartments had been depleted. Our results confirm that vesamicol inhibits the mobilization of transmitter from a reserve to a more readily releasable pool, and they also suggest that, under these experimental conditions, there might be some futile transmitter mobilization, apparently to sites other than nerve terminal active zones.     

Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. II. Effects of electrical stimulation and high potassium

Frog cutaneous pectoris nerve muscle preparations were studied by the freeze-fracture technique under the following conditions: (a) during repetitive indirect stimulation for 20 min, 10/s; (b) during recovery from this stimulation; and (c) during treatment with 20 mM K+. Indirect stimulation causes numerous dimples or protuberances to appear on the presynaptic membrane of nerve terminal, and most are located near the active zones. Deep infoldings of the axolemma often develop between the active zones. Neither the number nor the distribution of dimples, protuberances, of infoldings changes markedly during the first minute of recovery. The number of dimples, protuberances, and infoldings is greatly reduced after 10 min of recovery. Since endocytosis proceeds vigorously during the recovery periods, we conclude that endocytosis occurs mostly at the active zones, close to the sites of exocytosis. 20 mM K+ also causes many dimples or protuberances to appear on the axolemma of the nerve terminal but they are distributed almost uniformly along the presynaptic membrane. Experiments with horseradish peroxidase (HRP) show that recycling of synaptic vesicles occurs in 20 mM K+. This recycling is not accompanied by changes in the number of coated vesicles. Since both exocytosis and endocytosis occur in 20 mM K+, it is difficult to account for this unique distribution. However, we suggest that K+ causes dimples or protuberances to appear between the active zones because it activates latent sites of exocytosis specified by small numbers of large intramembrane particles located between active zones. The activation of latent release sites may be related to the complex effects that K+ has on the quantal release of neurotransmitter.     

Synaptic vesicle pools at the frog neuromuscular junction

We have characterized the morphological and functional properties of the readily releasable pool (RRP) and the reserve pool of synaptic vesicles in frog motor nerve terminals using fluorescence microscopy, electron microscopy, and electrophysiology. At rest, about 20% of vesicles reside in the RRP, which is depleted in about 10 s by high-frequency nerve stimulation (30 Hz); the RRP refills in about 1 min, and surprisingly, refilling occurs almost entirely by recycling, not mobilization from the reserve pool. The reserve pool is depleted during 30 Hz stimulation with a time constant of about 40 s, and it refills slowly (half-time about 8 min) as nascent vesicles bud from randomly distributed cisternae and surface membrane infoldings and enter vesicle clusters spaced at regular intervals along the terminal. Transmitter output during low-frequency stimulation (2-5 Hz) is maintained entirely by RRP recycling; few if any vesicles are mobilized from the reserve pool.     

Regulation of synaptic vesicles pools within motor nerve terminals during short-term facilitation and neuromodulation.

The reserve pool (RP) and readily releasable pool (RRP) of synaptic vesicles within presynaptic nerve terminals were physiologically differentiated into distinctly separate functional groups. This was accomplished in glutamatergic nerve terminals by blocking the glutamate transporter with dl-threo-beta-benzyloxyaspartate (TBOA; 10 microM) during electrical stimulation with either 40 Hz of 10 pulses within a train or 20- or 50-Hz continuous stimulation. The 50-Hz continuous stimulation decreased the excitatory postsynaptic potential amplitude 60 min faster than for the 20-Hz continuous stimulation in the presence of TBOA (P < 0.05). There was no significant difference between the train stimulation and 20-Hz continuous stimulation in the run-down time in the presence of TBOA. After TBOA-induced synaptic depression, the excitatory postsynaptic potentials were rapidly (<1 min) revitalized by exposure to serotonin (5-HT, 1 microM) in every preparation tested (P < 0.05). At this glutamatergic nerve terminal, 5-HT promotes an increase probability of vesicular docking and fusion. Quantal recordings made directly at nerve terminals revealed smaller quantal sizes with TBOA exposure with a marked increase in quantal size as well as a continual appearance of smaller quanta upon 5-HT treatment after TBOA-induced depression. Thus 5-HT was able to recruit vesicles from the RP that were not rapidly depleted by acute TBOA treatment and electrical stimulation. The results support the notion that the RRP is selectively activated during rapid electrical stimulation sparing the RP; however, the RP can be recruited by the neuromodulator 5-HT. This suggests at least two separate kinetic and distinct regulatory paths for vesicle recycling within the presynaptic nerve terminal.     

EVIDENCE FOR RECYCLING OF SYNAPTIC VESICLE MEMBRANE DURING TRANSMITTER RELEASE AT THE FROG NEUROMUSCULAR JUNCTION

When the nerves of isolated frog sartorius muscles were stimulated at 10 Hz, synaptic vesicles in the motor nerve terminals became transiently depleted. This depletion apparently resulted from a redistribution rather than disappearance of synaptic vesicle membrane, since the total amount of membrane comprising these nerve terminals remained constant during stimulation. At 1 min of stimulation, the 30% depletion in synaptic vesicle membrane was nearly balanced by an increase in plasma membrane, suggesting that vesicle membrane rapidly moved to the surface as it might if vesicles released their content of transmitter by exocytosis. After 15 min of stimulation, the 60% depletion of synaptic vesicle membrane was largely balanced by the appearance of numerous irregular membrane-walled cisternae inside the terminals, suggesting that vesicle membrane was retrieved from the surface as cisternae. When muscles were rested after 15 min of stimulation, cisternae disappeared and synaptic vesicles reappeared, suggesting that cisternae divided to form new synaptic vesicles so that the original vesicle membrane was now recycled into new synaptic vesicles. When muscles were soaked in horseradish peroxidase (HRP), this tracerfirst entered the cisternae which formed during stimulation and then entered a large proportion of the synaptic vesicles which reappeared during rest, strengthening the idea that synaptic vesicle membrane added to the surface was retrieved as cisternae which subsequently divided to form new vesicles. When muscles containing HRP in synaptic vesicles were washed to remove extracellular HRP and restimulated, HRP disappeared from vesicles without appearing in the new cisternae formed during the second stimulation, confirming that a one-way recycling of synaptic membrane, from the surface through cisternae to new vesicles, was occurring. Coated vesicles apparently represented the actual mechanism for retrieval of synaptic vesicle membrane from the plasma membrane, because during nerve stimulation they proliferated at regions of the nerve terminals covered by Schwann processes, took up peroxidase, and appeared in various stages of coalescence with cisternae. In contrast, synaptic vesicles did not appear to return directly from the surface to form cisternae, and cisternae themselves never appeared directly connected to the surface. Thus, during stimulation the intracellular compartments of this synapse change shape and take up extracellular protein in a manner which indicates that synaptic vesicle membrane added to the surface during exocytosis is retrieved by coated vesicles and recycled into new synaptic vesicles by way of intermediate cisternae.     

Acetylcholine compartments in mouse diaphragm. Comparison of the effects of black widow spider venom, electrical stimulation, and high concentrations of potassium

We have studied the effects of 25 mM potassium, electrical stimulation of the phrenic nerve, and crude black widow spider venom on the ultrastructure, electrophysiology, and acetylcholine (ACh) contents of mouse diaphragms. About 65% of the ACh in diaphragms is contained in a depletable store in the nerve terminals. The rest of the ACh is contained in a nondepletable store that may correspond to the store that remains in denervated muscles and includes, in addition, ACh in the intramuscular branches of the phrenic nerve. About 4% of the ACh released from the depletable store at rest is secreted as quanta and may come from the vesicles, while 96% is secreted in a nonquantized form and comes from an extravesicular pool. The size of the extravesicular pool is uncertain: it could be less than 10%, or as great as 50%, of the depletable store. K causes a highly (but perhaps not perfectly) selective increase in the rate of quantal secretion so that quanta account for about 50% of the total ACh released from K- treated diaphragms. K, or electrical stimulation of the phrenic nerve, depletes both the vesicular and extravesicular pools of ACh when hemicholinium no. 3 (HC-3) is present. However, most of the vesicles are retained under these conditions so that the diaphragms are able to increase slightly their rates of release of ACh when K is added. Venom depletes the terminals of their vesicles and abolishes the release of quanta of ACh. It depletes the vesicular pool of ACh (since it depletes the vesicles), but may only partially deplete the extravesicular pool (since it reduces resting release only 10--40%). The rate of release of ACh from the residual extravesicular pool does not increase when 25 mM K is added. Although we cannot exclude the possibility that stimulation may double the rate of release of ACh from the extravesicular pool, our results are compatible with the idea that the ACh released by stimulation comes mainly from the vesicles and that, when synthesis is inhibited by HC-3, ACh may be exchanged between the extravesicular pool and recycled vesicles.     

Redistribution of synaptophysin and synapsin I during alpha-latrotoxin- induced release of neurotransmitter at the neuromuscular junction

The distribution of two synaptic vesicle-specific phosphoproteins, synaptophysin and synapsin I, during intense quantal secretion was studied by applying an immunogold labeling technique to ultrathin frozen sections. In nerve-muscle preparations treated for 1 h with a low dose of alpha-latrotoxin in the absence of extracellular Ca2+ (a condition under which nerve terminals are depleted of both quanta of neurotransmitter and synaptic vesicles), the immunolabeling for both proteins was distributed along the axolemma. These findings indicate that, in the presence of a block of endocytosis, exocytosis leads to the permanent incorporation of the synaptic vesicle membrane into the axolemma and suggest that, under this condition, at least some of the synapsin I molecules remain associated with the vesicle membrane after fusion. When the same dose of alpha-latrotoxin was applied in the presence of extracellular Ca2+, the immunoreactivity patterns resembled those obtained in resting preparations: immunogold particles were selectively associated with the membrane of synaptic vesicles, whereas the axolemma was virtually unlabeled. Under this condition an active recycling of both quanta of neurotransmitter and vesicles operates. These findings indicate that the retrieval of components of the synaptic vesicle membrane is an efficient process that does not involve extensive intermixing between molecular components of the vesicle and plasma membrane, and show that synaptic vesicles that are rapidly recycling still have the bulk of synapsin I associated with their membrane.     

Stimulation-depletion of serotonin and noradrenaline from vesicles of sympathetic nerves in the pineal gland of the rat

Summary The pineal gland of the rat receives a rich nervous supply originating from the superior cervical ganglia. These fibers contain serotonin in addition to their neurotransmitter, noradrenaline. Cytochemical studies at the ultrastructural level have shown that both amines are present in the cores of the granular vesicles that are characteristic of these nerves. It is presently shown that the bilateral electrical stimulation of the preganglionic fibers innervating the ganglia markedly reduces the number of small sites reacting cytochemically for both noradrenaline and serotonin, these sites corresponding to the cores of small granular vesicles, while the larger reactive sites (cores of large vesicles) remain unaltered. The vesicles are retained in nerve terminals after stimulation, as observed in conventionally processed tissues, although with altered sizes and shapes. Apart from these cytochemical and structural changes, nerve stimulation also reduces the endogenous noradrenaline content of the pineal gland. Thus, both noradrenaline and serotonin are released from their storage sites in pineal sympathetic nerves after electrical stimulation in vivo. This suggests the possibility that several substances with presumed transmitter or modulatory functions might be simultaneously released by nerve impulses from a given nerve terminal.     

Does nerve impulse activity modulate fast axonal transport?

The possibility that the amount of newly synthesized material made available for fast axonal transport is regulated by nerve impulse activity was examined in an in vitro preparation of bullfrog dorsal root ganglia (DRG) and sciatic nerve. Under conditions that precluded effects of impulse activity on either uptake or incorporation of precursor, patterned stimulation of the sciatic nerve (1 out of every 2 s) produced a frequency- and time-dependent decrease in the amount of radiolabeled protein accumulating at a nerve ligature. The response to patterned stimulation was significantly greater than that to continuous stimulation when the same number of stimuli were delivered. In unligated nerve preparations, patterned stimulation decreased the amplitude of the transport profile with no concomitant change in the wave front distance. Nerve stimulation produced no observable ultrastructural alterations within neuronal cell bodies of the DRG. We propose that the physiological significance of these results is not that nerve impulse activity decreases fast axonal transport, but that the amount of transport increases during periods of electrical quiescence. According to this hypothesis, activity-dependent macromolecules of the axolemma and nerve terminals are replenished during periods when the neuron is firing less frequently. These findings are discussed in light of reports that chronic in vivo stimulation increases the amount of fast-transported, radiolabeled protein (Chan et al., 1989) and that TTX-blockade of neuronal activity has no effect on protein transport (Edwards and Grafstein, 1984; Riccio and Matthews, 1985).     

Acetylcholine Synthesis and Release by a Sympathetic Ganglion in the Presence of 2-(4-Phenylpiperidino) Cyclohexanol (AH5183)

These experiments measured the release and the synthesis of acetylcholine (ACh) by cat sympathetic ganglia in the presence of 2-(4-phenylpiperidino) cyclohexanol (AH5183), an agent that blocks the uptake of ACh into synaptic vesicles. Evoked transmitter release during short periods of preganglionic nerve stimulation was not affected by AH5183, but release during prolonged stimulation was not maintained in the drug's presence, whereas it was in the drug's absence. The amount of ACh releasable by nerve impulses in the presence of AH5183 was 194 +/- 10 pmol, which represented 14 +/- 1% of the tissue ACh store. The effect of AH5183 on ACh release was not well antagonized by 4-aminopyridine (4-AP), and not associated with inhibition of stimulation-induced calcium accumulation by nerve terminals. It is concluded that AH5183 blocks ACh release indirectly, and that the proportion of stored ACh releasable in the compound's presence represents transmitter in synaptic vesicles available to the release mechanism. The synthesis of ACh during 30 min preganglionic stimulation in the presence of AH5183 was 2,448 +/- 51 pmol and in its absence it was 2,547 +/- 273 pmol. Thus, as the drug decreased ACh release it increased tissue content. The increase in tissue content of ACh in the presence of AH5183 was not evident in resting ganglia; it was evident in stimulated ganglia whether or not tissue cholinesterase was inhibited; it was increased by 4-AP and reduced by divalent cation changes expected to decrease calcium influx during nerve terminal depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synaptophysin (p38) at the frog neuromuscular junction: its incorporation into the axolemma and recycling after intense quantal secretion

Recycling of synaptophysin (p38), a synaptic vesicle integral membrane protein, was studied by the use of antisera raised against the protein purified from frog brain. When frog cutaneous pectoris muscles were fixed at rest, a bright, specific immunofluorescent signal was observed in nerve-terminal regions only if their plasma membranes had been previously permeabilized. When muscles were fixed after they had been treated for 1 h with a low dose of alpha-latrotoxin in Ca2+-free medium, an equally intense fluorescence could be observed without previous permeabilization. Under this condition, alpha-latrotoxin depletes nerve terminals of their quantal store of acetylcholine and of synaptic vesicles. These results indicate that fusion of synaptic vesicles leads to the exposure of intravesicular antigenic determinants of synaptophysin on the outer surface of the axolemma, and provide direct support for the vesicle hypothesis of neurotransmitter release. After 1 h treatment with the same dose of alpha-latrotoxin in the presence of 1.8 mM extracellular Ca2+, immunofluorescent images were obtained only after permeabilization with detergents. Under this condition, the vesicle population was maintained by an active process of recycling and more than two times the initial store of quanta were secreted. Thus, despite the active turnover of synaptic vesicles and of quanta of neurotransmitter, no extensive intermixing occurs between components of the vesicle and presynaptic plasma membrane.     

Differentiation of Nerve Terminals in the Crayfish Opener Muscle and Its Functional Significance

Junctional potentials (jp's) recorded from superficial distal fibers of the crayfish opener muscle are up to 50 times larger than jp' in superficial central fibers when the single motor axon that innervates the muscle is stimulated at a frequency of 1/sec or less. At 80/sec, in contrast, central jp's are up to four times larger than those observed in distal fibers. The tension produced by single muscle fibers of either type is directly proportional to the integral of the time-voltage curve minus an excitation-contraction coupling threshold of 3 mv. Distal fibers therefore produce almost all the total muscle tension at low frequencies of stimulation and central fibers add an increasingly greater contribution as their nerve endings begin to facilitate in response to increased rate of motor discharge. Differentiation of muscle membrane characteristics (input resistance, space constant, time constant) cannot account for these differences in facilitation ratios. The mechanism of neuronal differentiation is not based upon the size or effectiveness of transmitter quanta, since equal sized jp's have equal variances;: mjp sizes and variances are also equal. No differences were found between fiber types in rates of transmitter mobilization, density of innervation, or the relationship between transmitter release and terminal depolarization. Single terminals on distal fibers were found to release transmitter with a greater probability than central terminals. More effective invasion of distal terminals by the nerve impulse at low frequencies can account for the difference.     

Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction

Frog nerve-muscle preparations were quick-frozen at various times after a single electrical stimulus in the presence of 4-aminopyridine (4-AP), after which motor nerve terminals were visualized by freeze-fracture. Previous studies have shown that such stimulation causes prompt discharge of 3,000-6,000 synaptic vesicles from each nerve terminal and, as a result, adds a large amount of synaptic vesicle membrane to its plasmalemma. In the current experiments, we sought to visualize the endocytic retrieval of this vesicle membrane back into the terminal, during the interval between 1 s and 2 min after stimulation. Two distinct types of endocytosis were observed. The first appeared to be rapid and nonselective. Within the first few seconds after stimulation, relatively large vacuoles (approximately 0.1 micron) pinched off from the plasma membrane, both near to and far away from the active zones. Previous thin-section studies have shown that such vacuoles are not coated with clathrin at any stage during their formation. The second endocytic process was slower and appeared to be selective, because it internalized large intramembrane particles. This process was manifest first by the formation of relatively small (approximately 0.05 micron) indentations in the plasma membrane, which occurred everywhere except at the active zones. These indentations first appeared at 1 s, reached a peak abundance of 5.5/micron2 by 30 s after the stimulus, and disappeared almost completely by 90 s. Previous thin-section studies indicate that these indentations correspond to clathrin-coated pits. Their total abundance is comparable with the number of vesicles that were discharged initially. These endocytic structures could be classified into four intermediate forms, whose relative abundance over time suggests that, at this type of nerve terminal, endocytosis of coated vesicles has the following characteristics: (a) the single endocytotic event is short lived relative to the time scale of two minutes; (b) earlier forms last longer than later forms; and (c) a single event spends a smaller portion of its lifetime in the flat configuration soon after the stimulus than it does later on.     

Changes in cholinergic synaptic vesicle populations and the ultrastructure of the nerve terminal membranes of Narcine brasiliensis electron organ stimulated to fatigue in vivo

Narcine brasiliensis electric organ was stimulated to fatigue in vivo. Electrical display of organ output and biochemical assay of bound acetylcholine (ACh) and ATP in isolated vesicles were used to assess the state of fatigue relative to denervated control organs of the same fish. A morphometric analysis of the fate of the synaptic vesicle populations in the nerve terminals was carried out. Statistically significant morphological changes in vesicle populations and plasma membranes were observed between control and fatigued electroplaque stacks from individual fish. Pooled data from several fish were used to evaluate the possible role of the different vesicle types in neurotransmission. Fatigue resulted in the loss of 49% of the total vesicle population and a 76% loss of vesicles with bound calcium (Ca). An approximately equivalent increase in the nerve-terminal plasma membrane area was measured. This was predominantly in the form of fingerlike protrusions and/or invaginations of the terminals which were present in the control organs but which were significantly increased by stimulation. Vesicle attachments to the nerve terminal membrane were reduced by 90%. This suggests that the failure in transmission may be due to reduction in the number of vesicles which are loaded with transmitter and can attach to the terminal membrane. The Ca-binding capacity of the lost vesicles was not transferred to the plasma membranes. This result was interpreted as support for the hypothesis that vesicle-bound ATP provides the Ca-binding site.     

Effects of high calcium solutions on glutamate sensitivity of crayfish muscle fibres

Crayfish neuromuscular preparations were studied after 18--36 h exposure to high calcium solutions. As previously reported for frog neuromuscular preparations the treatment damaged the nerve terminals and decreased junctional potentials. The resting potentials and input resistances of the muscle fibres were not affected; but their sensitivity to glutamate was significantly decreased when compared to that of control muscles. After exposure to high calcium, the sensitivity to gamma-aminobutyric acid, the putative transmitter at inhibitory synapses, was increased. Apparently normal twitches were elicited by direct stimulation, and calcium spikes could still be observed in the fibres. A decreased sensitivity to glutamate was also noted in experiments carried out on denervated muscles 8 months after section of the motor axons. Possible relations between nerve terminal damage and the decrease in sensitivity to glutamate are discussed.     

The fusogenic substance dimethyl sulfoxide enhances exocytosis in motor nerve endings

Fusion of synaptic vesicles with the surface membrane of the nerve terminal is a key step in synaptic transmission, which normally requires the entry of calcium ions into the cell. We report that this fusion and the subsequent liberation of transmitter can also be induced by the fusogenic substances DMSO (dimethyl sulfoxide) and PEG (poly(ethylene glycol)). Calcium ions and DMSO exhibit a synergistic effect in the fusion of synaptic vesicles with the axolemma, resembling their action on fusion phenomena in liposomes.     

Activity-Dependent Bulk Synaptic Vesicle Endocytosis—A Fast,High Capacity Membrane Retrieval Mechanism

Central nerve terminals are placed under considerable stress during intense stimulation due to large numbers of synaptic vesicles (SVs) fusing with the plasma membrane. Classical clathrin-dependent SV endocytosis cannot correct for the large increase in nerve terminal surface area in the short term, due to its slow kinetics and low capacity. During such intense stimulation, an additional SV retrieval pathway is recruited called bulk endocytosis. Recent studies have shown that bulk endocytosis fulfils all of the physiological requirements to remedy the acute changes in nerve terminal surface area to allow the nerve terminal to continue to function. This review will summarise the recent developments in the field that characterise the physiology of bulk endocytosis which show that it is a fast, activity-dependent and high capacity mechanism that is essential for the function of central nerve terminals.     

Increased acetylcholine synthesis and release following presynaptic activity in a sympathetic ganglion

Abstract: The acetylcholine (ACh) content of sympathetic ganglia increases above its normal level following a period of preganglionic nerve stimulation. In the present experiments, this extra ACh that accumulates following activity was labeled radioactively from [³H]choline and its specific activity was compared with that of ACh subsequently released during preganglionic nerve stimulation. The specific activity of the released ACh was similar to that of the total tissue ACh, suggesting that the extra ACh mixes fully with endogenous stores. The present experiments also show that transmitter release during neuronal stimulation is necessary for the poststimulation increase in transmitter store. However, the increase was not evident when transmitter release was induced by K⁺. It is concluded that both transmitter release and impulse invasion of the nerve terminals are necessary for the adaptive phenomenon to manifest itself. The role of choline delivery and choline acetyltransferase activity in generating the poststimulation increase in transmitter store was tested. When choline transport activity measured as choline analogue (homocholine) accumulation increased, ACh synthesis was increased and when transport activity was not increased, neither was ACh synthesis. There was no poststimulation increase in measured choline acetyltransferase activity.     

Normal biogenesis and cycling of empty synaptic vesicles in dopamine neurons of vesicular monoamine transporter 2 knockout mice

The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging dopamine and other monoamines into synaptic vesicles and thereby plays an essential role in dopamine neurotransmission. Dopamine neurons in mice lacking VMAT2 are unable to store or release dopamine from their synaptic vesicles. To determine how VMAT2-mediated filling influences synaptic vesicle morphology and function, we examined dopamine terminals from VMAT2 knockout mice. In contrast to the abnormalities reported in glutamatergic terminals of mice lacking VGLUT1, the corresponding vesicular transporter for glutamate, we found that the ultrastructure of dopamine terminals and synaptic vesicles in VMAT2 knockout mice were indistinguishable from wild type. Using the activity-dependent dyes FM1-43 and FM2-10, we also found that synaptic vesicles in dopamine neurons lacking VMAT2 undergo endocytosis and exocytosis with kinetics identical to those seen in wild-type neurons. Together, these results demonstrate that dopamine synaptic vesicle biogenesis and cycling are independent of vesicle filling with transmitter. By demonstrating that such empty synaptic vesicles can cycle at the nerve terminal, our study suggests that physiological changes in VMAT2 levels or trafficking at the synapse may regulate dopamine release by altering the ratio of fillable-to-empty synaptic vesicles, as both continue to cycle in response to neural activity.