Assembly of SNARE Core Complexes Prior to Neurotransmitter Release Sets the Readily Releasable Pool of Synaptic Vesicles *

Precise regulation of neurotransmitter release is essential for the normal function of neural networks, but the mechanisms involved are largely unclear. Using superfused synaptosomes, we have studied the readily releasable pool of synaptic vesicles, measured as the amount of release triggered by hypertonic sucrose. We show that activation of presynaptic metabotropic glutamate receptors by dihydroxyphenylglycine and stimulation of protein kinase C by phorbol esters enhance the readily releasable pool of glutamate. Although the molecular nature of the readily releasable pool is unknown, one possibility is that during its generation, SNARE proteins form full core complexes, and that core complex formation occurs prior to neurotransmitter release. To test this possibility, we employed N-ethylmaleimide (NEM), an inhibitor of the ATPase N-ethylmaleimidesensitive factor that dissociates core complexes, to study the relation of the readily releasable pool to core complex assembly in synaptosomes. NEM induced a dosedependent increase in the readily releasable pool of neurotransmitters but by itself did not trigger release. Direct measurements of core complexes confirmed that NEM caused an increase in the levels of SNARE core complexes under these conditions. Our data suggest that in the readily releasable pool of synaptic vesicles, SNARE proteins are fully assembled into core complexes, and that SNARE complex assembly is a target of presynaptic regulation.

Neurotransmitter release is mediated by synaptic vesicle exocytosis at the active zone of a synapse.Most synapses contain a pool of docked vesicles at the active zone that can be stimulated for rapid release; this pool is referred to as the readily releasable pool (1)(2)(3)(4)(5)(6).Hypertonic solutions, such as 0.5 M sucrose, trigger synaptic vesicle exocytosis from presynaptic nerve terminals by an unknown, Ca 2ϩ -independent mechanism (7).Hypertonic sucrose and strong depolarizations stimulate release of the same vesicle pool that is thought to represent the readily releasable pool (8,9).The size of the readily releasable pool correlates with the probability of release (10 -12) and is up-regulated by protein kinase C (13) or downregulated during long term depression (14).Despite an exten-sive electrophysiological characterization, however, little is known about the biochemical nature of the readily releasable pool.
Exocytosis of synaptic vesicles involves the assembly of core complexes from SNARE proteins.Core complexes are formed by the synaptic vesicle SNARE protein synaptobrevin-vesicleassociated membrane protein and the plasma membrane SNARE proteins syntaxin and SNAP-25 (15).The synaptic core complex is composed of a four-helix bundle that is very stable and resistant to SDS denaturation (16,17; reviewed in Refs.18 and 19).Assembled core complexes are dissociated by a specific ATPase, N-ethylmaleimide-sensitive factor (NSF), 1 which is inhibited by NEM (15).The first clue to the function of the core complex came from the discovery that in presynaptic nerve terminals, SNAREs are the targets for botulinum and tetanus toxins (for reviews, see Refs.20 and 21).These toxins selectively inhibit synaptic vesicle fusion but do not interfere with the attachment of synaptic vesicles to the active zone.The toxins inhibit fusion independent of whether it is triggered physiologically by Ca 2ϩ or artificially by hypertonic sucrose, suggesting a central function for SNAREs in fusion, although their precise role has remained elusive.
In the current study, we have examined the role of SNARE core complexes in the regulation of the readily releasable pool of synaptic vesicles.For this purpose we used synaptosomes, a biochemical system that allows exploring multiple neurotransmitters and correlating release with the biochemical properties of the presynaptic terminal (9,22).Our results reveal that PKC up-regulates the readily releasable pool of glutamate and norepinephrine, but not of GABA.A similar up-regulation of the readily releasable pool can be achieved non-physiologically by NEM, which increases core complex abundance and bypasses the PKC step, and also acts on GABAergic synapses.The data uncover distinct pathways in the regulation of release for different neurotransmitters and suggest that as a general mechanism, the readily releasable pool corresponds to vesicles with assembled core complexes.
Measurement of 3  Analysis of Core Complexes-Fresh synaptosomes were incubated for 5 min at 35 °C in aerated KBH buffer, followed by a 10-min incubation in KBH buffer with NEM.Afterward, synaptosomes were dissolved in SDS-PAGE sample buffer and immediately analyzed by SDS-PAGE with or without boiling, followed by immunoblotting using mono-and polyclonal antibodies to SNAP-25, syntaxin, and synaptobrevin (9).To analyze the core complex by cross-linking, DSP was selected from 8 screened cross-linking agents, because it resulted in the clearest crosslinking of the core complex in synaptosomes.Synaptosomes were treated as above except that 0.3-1.0 mM DSP were added directly to intact synaptosomes during the last 5 min of incubation.Cross-linking was quenched by adding 0.2 volumes of 1.5 M Tris-HCl, pH 6.8, containing 6% SDS.Samples were boiled for 5 min prior to immunoprecipitation with polyclonal syntaxin (I378) or SNAP-25 antibodies (I733) as described (25).Immunoprecipitates were analyzed by SDS-PAGE without reducing agents and by immunoblotting.Immunoblots were reacted with 125 I-labeled secondary antibodies, and the 125 I signal was quantified in a phosphoimager using a rectangle drawn across the entire width of the gel at the level of the core complex and of the monomer.An automatic integration method was then used to calculate the relative intensity of each band for a given lane to exclude observer bias.To ensure that the 80-kDa band observed corresponds to crosslinked core complex, samples were run in the presence of reducing agents (100 mM dithiothreitol), which cleave the cross-linker DSP and abolished the core complex band.To ensure linearity of the immunoblotting signal, we confirmed that amounts of 3 to 30 g of protein per lane resulted in linear signals.

Regulation of the Readily Releasable Glutamate Pool by
Presynaptic Glutamate Receptors-In synaptosomes, activation of type I presynaptic glutamate receptors has been reported to up-regulate glutamate release in a PKC-dependent manner by enhancing Ca 2ϩ influx during KCl depolarization (27,28).However, in cultured hippocampal neurons activation of PKC increases release by augmenting the readily releasable pool of glutamate in a process that does not involve Ca 2ϩ influx (13).
To clarify this issue, we investigated the effect of DHPG, an agonist for type I metabotropic glutamate receptors, on glutamate release from synaptosomes.Release was triggered in parallel either by KCl depolarization in the presence of Ca 2ϩ or by hypertonic sucrose in the absence of Ca 2ϩ (Fig. 1).DHPG had no significant effect on basal glutamate efflux but enhanced subsequent glutamate exocytosis evoked either by KCl depolarization or by hypertonic sucrose.The effect was not smaller for Ca 2ϩ -independent sucrose-evoked release than for Ca 2ϩ -dependent KCl-evoked release, suggesting that changes in Ca 2ϩ -channel properties are not a major factor in the potentiation of release and that activation of type I metabotropic glutamate receptors enhances the pool of readily releasable synaptic vesicles.
PKC Up-regulates the Readily Releasable Pool of Vesicles-Type I metabotropic glutamate receptors stimulate phospholipase C activity, which in turn activates PKC via production of diacylglycerol.Therefore we investigated whether PKC is involved in the up-regulation of the readily releasable pool observed upon DHPG treatment by directly stimulating PKC in synaptosomes with the phorbol ester phorbol 12,13-dibutyrate (PDBu) prior to triggering secretion with hypertonic sucrose.In these experiments, all manipulations (phorbol ester treatments, washes, and stimulation of release by hypertonic sucrose) were performed in the absence of extracellular Ca 2ϩ to exclude the possibility that phorbol esters may cause Ca 2ϩ influx and thereby exert an indirect effect.
PDBu by itself did not change basal glutamate efflux (Fig. 2,  A and B).PDBu treatment did, however, result in a strong enhancement of sucrose-triggered release; the enhancement was inhibited by the specific PKC inhibitor bisindolylmaleimide (29).Like PDBu, bisindolylamide had no effect on baseline glutamate release.An enhancement of secretion similar to that obtained with PDBu was also observed with the related phorbol ester phorbol 12-myristate 13-acetate (1 M), which increased sucrose-evoked secretion by 232 Ϯ 34% (n ϭ 3; data not shown).The potentiation of the readily releasable pool by PDBu was dose-dependent and could not be obtained with an inactive isomer (␣-PDBu) (Fig. 2C).When we tested whether PDBu generally increased release from all synapses, we observed a strong enhancement of the secretion of another neurotransmitter, norepinephrine, whereas PDBu had no effect on the secretion of GABA (Fig. 3).The lack of effect on GABA release and the similar pharmacological findings in electrophysiological recordings (13) indicate that the effects of PDBu are specific and suggest differences in the molecular basis of the regulation of inhibitory and excitatory synapses.This result also provides independent support for the validity of the synaptosomal system in measuring the readily releasable pool of synaptic vesicles.
Effects of NEM on the Readily Releasable Pool of Vesicles and SNARE Core Complexes-What is the mechanism by which PKC stimulates the readily releasable pool of synaptic vesicles?During synaptic vesicles exocytosis, the vesicular SNARE synaptobrevin-vesicle-associated membrane protein assembles into core complexes with the plasma membrane SNAREs syntaxin and SNAP-25 (15).The core complexes are then dissociated again, either before or after fusion, by the ATPase NSF, which can be inhibited with the nonspecific sulfhydryl agent NEM.It is unknown precisely when SNARE core complex assembly occurs in exocytosis and whether it irreversibly leads to fusion.One possibility is that the readily releasable pool corresponds to a partially fused vesicle population that may have already fully assembled SNARE core complexes (18).If this was correct, enhancing the abundance of assembled core complexes should increase the size of the readily releasable pool.
To test this hypothesis, we treated synaptosomes with NEM at several concentrations, washed the synaptosomes, and measured the effect of NEM treatment on the size of the readily releasable pool monitored as the amount of release evoked by hypertonic sucrose (Fig. 4).As in the phorbol-ester experiments, all manipulations were performed in the absence of extracellular Ca 2ϩ to exclude confounding effects of artifactual Ca 2ϩ influx.Low concentrations of NEM had no acute effect on neurotransmitter efflux from the superfused synaptosomes, and only high NEM concentrations (100 M) caused leakage, presumably because NEM is cytotoxic at this concentration.In the washing period after NEM application, baseline release was normal for all samples.However, when we subsequently triggered release with hypertonic sucrose, we observed a major enhancement of release as a function of NEM (Fig. 4).This increase was observed for all three neurotransmitters tested (glutamate, GABA, and norepinephrine) in contrast to phorbol esters, which were inactive for GABA (Fig. 3).
NEM Potentiates Core Complex Abundance-One possible cause for the NEM-dependent increase in the readily releasable pool is that in resting nerve terminals, core complexes assemble and dissociate in a dynamic equilibrium without re-

FIG. 3. Phorbol esters enhance the readily releasable pool of norepinephrine (NE) but not GABA.
A and B, representative experiments of GABA or norepinephrine (NE) release from synaptosomes during application of PDBu or Me 2 SO, followed by stimulation with a 30-s pulse of hypertonic sucrose.C, summary graph of multiple experiments examining GABA and norepinephrine secretion during treatment with 1 M PDBu, after treatment during the washout period (Basal), and during hypertonic sucrose stimulation (Sucrose).Note that PDBu by itself does not evoke secretion in either system and potentiates only sucrose-evoked release of norepinephrine (asterisk ϭ p Ͻ 0.05; n ϭ 3-4).lease of neurotransmitters.NEM would then disrupt this equilibrium by inhibiting core complex disassembly and thereby force vesicles into a state of assembled core complexes that increase the readily releasable pool of vesicles.To test this hypothesis, we studied the abundance of core complexes in synaptosomes as a function of NEM treatment.Synaptosomes were exposed to NEM for 10 min, with a chemical cross-linking agent (DSP) present during the last 5 min of treatment.The reaction was then stopped by addition of 6% SDS in 1.5 M Tris-HCl, and the samples were boiled and immunoprecipitated with polyclonal antibodies to syntaxin (I378) or SNAP-25 (I733).Cross-linked core complexes were analyzed by SDS-PAGE and immunoblotting with monoclonal antibodies, 125 Ilabeled secondary antibodies, and phosphoimager quantification.A dose-dependent increase in the signal of the core complex at 80 kDa was observed (Fig. 5A).Control experiments in which the samples were reduced prior to SDS-PAGE to cleave the cross-linker revealed that the 80-kDa band corresponds to a chemically cross-linked protein complex, and analysis of the cut-out 80-kDa band confirmed that it contains all three SNAREs (Ref.9 and data not shown).Quantitation of the core complex signal confirmed that treatment of synaptosomes with NEM (10 -100 M) increased the steady-state abundance of core complexes in a dose-dependent fashion (Fig. 5B) that approximately corresponded to the increase in the readily releasable pool observed under the same conditions (Fig. 4).

DISCUSSION
Using synaptosomes, we have examined the relation between neurotransmitter release and the assembly of core complexes from synaptic SNARE proteins.We showed that treatments of synaptosomes with NEM or phorbol esters do not by themselves trigger neurotransmitter release but increase the size of the readily releasable pool of synaptic vesicles available for subsequent release.This effect is likely to be physiologically important, because a similar increase in the readily releasable pool can be obtained upon stimulation of presynaptic metabotropic glutamate receptors.The most surprising result of our study, however, was the enhancement of the readily releasable pool by NEM, an inhibitor of the ATPase NSF.Although NEM is a rather toxic sulfhydryl reagent, it did not damage synaptosomes at low doses, for example by causing transmitter leakage or unresponsiveness to stimulation.Instead, low doses of NEM increased sucrose-evoked release of all neurotransmitters tested (glutamate, norepinephrine, and GABA).Further- more, direct measurements showed that NEM increased the abundance of SNARE core complexes in synaptosomes.These data provide evidence that the assembly of SNARE complexes at the synapse is dynamic and regulates the amount of vesicles to undergo exocytosis.Based on this observation, we suggest as a working hypothesis that core complexes are assembled from SNAREs prior to fusion pore opening and that the degree of assembly determines the size of the readily releasable pool (see model in Fig. 6).One possible mechanism that may underlie this process is that core complex assembly drives hemifusion as the physical correlate of the functionally defined "prefusion" reaction (18).
Our findings strongly agree with recent results obtained with an in vitro fusion system using yeast vacuoles (30).These experiments showed that SNARE core complexes, although required for fusion, can be disassembled before a full fusion pore is formed.In chromaffin cells the use of a SNAP-25 antibody also revealed that core complex formation and fusion are temporarily separate events (31).However, results from other systems appear to be more difficult to reconcile with our hypothesis.For example, fusion in permeabilized neuroendocrine cells is inhibited by botulinum and tetanus toxins at a late stage (32,33).Because the assembled core complex is resistant to toxin cleavage (16), this result could be interpreted to mean that core complex assembly occurs very late in fusion.However, in neuroendocrine cells only a small percentage of vesicles are prefused; these prefused vesicles would be largely invisible in the biochemical assays used.Because fusion is fast, assembly of core complexes and Ca 2ϩ triggering of fusion could easily occur during the time of the biochemical assay.Furthermore, the dynamic nature of the core complex, as observed in our NEM experiments, may allow cleavage even after initial core complexes have formed.Finally, the requirement of Ca 2ϩ for core complex formation in permeabilized neuroendocrine cells (33) could be due to the known role of Ca 2ϩ in the mobilization of secretory vesicles from the cytoskeleton (34,35).
Our findings support the idea that core complex assembly represents an important point of regulation in neurotransmitter secretion and that presynaptic plasticity can operate at the step of prefusion by regulating the size of the readily releasable pool.The effects of the activation of type I presynaptic glutamate receptors on the readily releasable pool suggest a positive feedback loop; secreted glutamate activates presynaptic receptors, which then increase the recruitment of vesicles for further exocytosis.Other presynaptic receptors, such as type III metabotropic glutamate receptors, may have opposing physiological effects (for example see Refs.36 and 37).The finding that PKC inhibitors abolish the increase in the readily releasable pool induced by phorbol esters indicates that PKC may be a regulatory pathway for the readily releasable pool size.Although the molecular target of PKC is currently unknown, it is possible that PKC modulates NSF activity and thereby regulates core complex assembly or that phosphorylation of munc18 is involved (38,39).Independent of what the molecular target for PKC is, our results suggest that a hierarchy of regulatory events operating at distinct steps (prefusion and Ca 2ϩ -triggered fusion pore opening) modulate synaptic exocytosis in a manner that contributes to the extraordinary precision of synaptic transmission.

FIG. 1 .
FIG. 1.The presynaptic glutamate-receptor agonist DHPG potentiates glutamate secretion by increasing the readily releasable pool.Synaptosomes loaded with 3 H-labeled glutamate and treated with 30 M DHPG or control buffer were stimulated in a superfusion chamber with 30-s pulses of 25 mM KCl (left) or 0.5 M sucrose (right).DHPG and control treatments were performed in Ca 2ϩcontaining medium whereas evoked release was triggered either in Ca 2ϩ -containing (KCl) or Ca 2ϩ -free (Sucrose) medium.Glutamate release was continuously measured in the superfusate as a function of treatment, as shown in a representative experiment in the top panels (A and C).The bottom panels (B and D) show summary graphs of multiple experiments in which basal glutamate release under control conditions was set to 100%, and all other secretion is expressed in % of basal release.Data shown are means Ϯ S.E., with asterisks indicating statistically significant differences of treated versus control samples (p Ͻ 0.05, n ϭ 7-8).

FIG. 2 .
FIG. 2. Phorbol esters enhance the readily releasable pool of glutamate by a PKC-dependent mechanism.A, representative experiment of glutamate secretion from synaptosomes during treatment with PDBu or vehicle (Me 2 SO), followed by stimulation with a 30-s pulse of hypertonic sucrose.B, summary graph of multiple experiments in which glutamate secretion from superfused synaptosomes was measured during PDBu treatment, under basal conditions following treatment, and during sucrose stimulation.Synaptosomes were either treated with PDBu alone (left) or with PDBu and the specific PKC inhibitor bisindolylmaleimide (1 M; right).Note that PDBu treatment by itself elicits no secretion.C, summary graph of experiments examining the effects of different concentrations of PDBu or its inactive analog ␣-phorbol 12,13-dibutyrate (␣-PDBu) on glutamate secretion evoked by hypertonic sucrose (n ϭ 3-7).

FIG. 4 .
FIG. 4. NEM increases the readily releasable pool of neurotransmitters (glutamate, norepinephrine, and GABA).A, representative experiment of glutamate release from superfused synaptosomes.Under constant superfusion with oxygenated Ca 2ϩ--free medium, synaptosomes were exposed to the indicated concentrations of NEM or control buffer for 5 min, washed for 2 min, and then stimulated for 30 s with 0.5 M sucrose.Released glutamate was measured in the perfusate.B, summary graphs from multiple experiments (n ϭ 3-7) of glutamate release from synaptosomes.Release evoked acutely by NEM treatment (left section, NEM), release after the wash-out period (center section, Basal), and the amount of release subsequently triggered by 0.5 M sucrose (right section; Sucrose) were determined as a function of NEM treatment.C, summary graphs of GABA and norepinephrine release experiments similar to those reported in A and B for glutamate.Data shown are means Ϯ S.E., with asterisks indicating statistically significant differences of treated versus control samples (p Ͻ 0.05).
, synaptosomes were incubated for 5 min at 33 °C in KBH buffer with 0.2 M 3 H-glutamate (24 Ci/mmol specific activity), 0.05 M 3 H-GABA (89 Ci/mmol), or 0.4 M 3 H-norepinephrine (12 Ci/mmol) together with 10 M pargyline and 0.6 mM ascorbic acid.Labeled synaptosomes were trapped on a glass fiber filter (GF/B) in a superfusion chamber and superfused at 33 °C and 0.8 ml/min with aerated (95% O 2 /5% CO 2 ) KBH buffer.To wash the synaptosomes and to apply various pharmacological agents, synaptosomes were super- H-Neurotransmitter Secretion-To label the releas-able neurotransmitter pool