The quantal release of glutamate depends on its transport into synaptic vesicles. Recent work has shown that a protein previously implicated in the uptake of inorganic phosphate across the plasma membrane catalyzes glutamate uptake by synaptic vesicles. However, only a subset of glutamate neurons expresses this vesicular glutamate transporter (VGLUT1). We now report that excitatory neurons lacking VGLUT1 express a closely related protein that has also been implicated in phosphate transport. Like VGLUT1, this protein localizes to synaptic vesicles and functions as a vesicular glutamate transporter (VGLUT2). The complementary expression of VGLUT1 and 2 defines two distinct classes of excitatory synapse.

Exocytosis of synaptic vesicles requires the formation of a fusion complex consisting of the synaptic vesicle protein synaptobrevin (vesicle-associated membrane protein, or VAMP) and the plasma membrane proteins syntaxin and soluble synaptosomal-associated protein of 25 kDa (or SNAP 25). In search of mechanisms that regulate the assembly of the fusion complex, it was found that synaptobrevin also binds to the vesicle protein synaptophysin and that synaptophysin-bound synaptobrevin cannot enter the fusion complex. Using a combination of immunoprecipitation, cross-linking, and in vitro interaction experiments, we report here that the synaptophysin-synaptobrevin complex is upregulated during neuronal development. In embryonic rat brain, the complex is not detectable, although synaptophysin and synaptobrevin are expressed and are localized to the same nerve terminals and to the same pool of vesicles. In contrast, the ability of synaptobrevin to participate in the fusion complex is detectable as early as embryonic day 14. The binding of synaptoporin, a closely related homolog of synaptophysin, to synaptobrevin changes in a similar manner during development. Recombinant synaptobrevin binds to synaptophysin derived from adult brain extracts but not to that derived from embryonic brain extracts. Furthermore, the soluble cytosol fraction of adult, but not of embryonic, synaptosomes contains a protein that induces synaptophysin-synaptobrevin complex formation in embryonic vesicle fractions. We conclude that complex formation is regulated during development and is mediated by a posttranslational modification of synaptophysin. Furthermore, we propose that the synaptophysin-synaptobrevin complex is not essential for exocytosis but rather provides a reserve pool of synaptobrevin for exocytosis that can be readily recruited during periods of high synaptic activity.

The segregation between vesicular glutamate and GABA storage and release forms the molecular foundation between excitatory and inhibitory neurons and guarantees the precise function of neuronal networks. Using immunoisolation of synaptic vesicles, we now show that VGLUT2 and VGAT, and also VGLUT1 and VGLUT2, coexist in a sizeable pool of vesicles. VGAT immunoisolates transport glutamate in addition to GABA. Furthermore, VGLUT activity enhances uptake of GABA and monoamines. Postembedding immunogold double labeling revealed that VGLUT1, VGLUT2, and VGAT coexist in mossy fiber terminals of the hippocampal CA3 area. Similarly, cerebellar mossy fiber terminals harbor VGLUT1, VGLUT2, and VGAT, while parallel and climbing fiber terminals exclusively contain VGLUT1 or VGLUT2, respectively. VGLUT2 was also observed in cerebellar GABAergic basket cells terminals. We conclude that the synaptic coexistence of vesicular glutamate and GABA transporters allows for corelease of both glutamate and GABA from selected nerve terminals, which may prevent systemic overexcitability by downregulating synaptic activity. Furthermore, our data suggest that VGLUT enhances transmitter storage in nonglutamatergic neurons. Thus, synaptic and vesicular coexistence of VGLUT and VGAT is more widespread than previously anticipated, putatively influencing fine-tuning and control of synaptic plasticity.

The brain adapts to experience by changing synaptic efficacy. Extensively studied mechanisms of plasticity include changes in the probability of transmitter release and in postsynaptic responsiveness. However, evidence from aminergic synapses indicates that the biochemistry of neurotransmitters provides an additional tool to alter synaptic strength. For example, dopaminergic transmission can be boosted by the precursor L-DOPA, which enhances the transmitter content of individual vesicles (Pothos et al. 1996) and counteracts Parkinson's disease.A similar principle may apply to one of the major central transmitters, the inhibitory amino acid GABA (y-aminobutyric acid). Drugs enhancing the tissue content of GABA act as anticonvulsants (Löscher et al. 1989; Taylor et al. 1992), suggesting that inhibitory tone depends on GABA supply. Moreover, the production of endogenous GABA varies, depending on the functional needs of the network: gonadotropin secretion from hypothalamic neurons is regulated through the negative feedback of gonadal steroids on the activity of the GABAproducing enzyme glutamate decarboxylase (GAD; Grattan et al. 1996); in hippocampal interneurons, suppression of GAD expression by oestradiol results in decreased inhibition and subsequent changes in dendritic spine 1. The production of the central inhibitory transmitter GABA (y-aminobutyric acid) varies in response to different patterns of activity. It therefore seems possible that GABA metabolism can determine inhibitory synaptic strength and that presynaptic GABA content is a regulated parameter for synaptic plasticity.2. We altered presynaptic GABA metabolism in cultured rat hippocampal slices using pharmacological tools. Degradation of GABA by GABA-transaminase (GABA-T) was blocked by y-vinyl-GABA (GVG) and synthesis of GABA through glutamate decarboxylase (GAD) was suppressed with 3-mercaptopropionic acid (MPA). We measured miniature GABAergic postsynaptic currents (mIPSCs) in CA3 pyramidal cells using the whole-cell patch clamp technique.3. Elevated intra-synaptic GABA levels after block of GABA-T resulted in increased mIPSC amplitude and frequency. In addition, tonic GABAergic background noise was enhanced by GVG. Electron micrographs from inhibitory synapses identified by immunogold staining for GABA confirmed the enhanced GABA content but revealed no further morphological alterations.4. The suppression of GABA synthesis by MPA had opposite functional consequences: mIPSC amplitude and frequency decreased and current noise was reduced compared with control. However, we were unable to demonstrate the decreased GABA content in biochemical analyses of whole slices or in electron micrographs.5. We conclude that the transmitter content of GABAergic vesicles is variable and that postsynaptic receptors are usually not saturated, leaving room for up-regulation of inhibitory synaptic strength. Our data reveal a new mechanism of plasticity at central inhibitory synapses and provide a rationale for the activity-dependent regulation of GABA synthesis in ...

Monoamines such as noradrenaline and serotonin are stored in secretory vesicles and released by exocytosis. Two related monoamine transporters, VMAT1 and VMAT2, mediate vesicular transmitter uptake. Previously we have reported that in the rat pheochromocytoma cell line PC 12 VMAT1, localized to peptide-containing secretory granules, is controlled by the heterotrimeric G-protein Go(2). We now show that in BON cells, a human serotonergic neuroendocrine cell line derived from a pancreatic tumor expressing both transporters on large, dense-core vesicles, VMAT2 is even more sensitive to G-protein regulation than VMAT1. The activity of both transporters is only downregulated by Galphao(2), whereas comparable concentrations of Galphao(1) are without effect. In serotonergic raphe neurons in primary culture VMAT2 is also downregulated by pertussis toxin-sensitive Go(2). By electron microscopic analysis from prefrontal cortex we show that VMAT2 and Galphao(2) associate preferentially to locally recycling small synaptic vesicles in serotonergic terminals. In addition, Go(2)-dependent modulation of VMAT2 also works when using a crude synaptic vesicle preparation from this brain area. We conclude that regulation of monoamine uptake by the heterotrimeric G proteins is a general feature of monoaminergic neurons that controls the content of both large, dense-core and small synaptic vesicles.

Variations in the neurotransmitter content of secretory vesicles enable neurons to adapt to network changes. Vesicular content may be modulated by vesicle-associated Go 2 , which down-regulates the activity of the vesicular monoamine transmitter transporters VMAT1 in neuroendocrine cells and VMAT2 in neurons. Blood platelets resemble serotonergic neurons with respect to transmitter storage and release. In streptolysin O-permeabilized platelets, VMAT2 activity is also downregulated by the G protein activator guanosine 5-(␤ i ␥-imido)triphosphate (GMppNp). Using serotonindepleted platelets from peripheral tryptophan hydroxylase knockout (Tph1؊/؊) mice, we show here that the vesicular filling initiates the G protein-mediated downregulation of VMAT2 activity. GMppNp did not influence VMAT2 activity in naive platelets from Tph1؊/؊ mice. GMppNp-mediated inhibition could be reconstituted, however, when preloading Tph1؊/؊ platelets with serotonin or noradrenaline. G␣ q mediates the down-regulation of VMAT2 activity as revealed from uptake studies performed with platelets from G␣ q deletion mutants. Serotonergic, noradrenergic, as well as thromboxane A 2 receptors are not directly involved in the down-regulation of VMAT2 activity. It is concluded that in platelets the vesicle itself regulates transmitter transporter activity via its content and vesicle-associated G␣ q .

The activity of vesicular monoamine transporters (VMATs) is down-regulated by the G-protein ␣-subunits of G o2 and G q , but the signaling pathways are not known. We show here that no such regulation is observed when VMAT1 or VMAT2 are expressed in Chinese hamster ovary (CHO) cells. However, when the intracellular compartments of VMAT-expressing CHO cells are preloaded with different monoamines, transport becomes susceptible to G-protein-dependent regulation, with differences between the two transporter isoforms. Epinephrine induces G-protein-mediated inhibition of transmitter uptake in CHOVMAT1 cells but prevents inhibition induced by dopamine in CHOVMAT2 cells. Epinephrine also antagonizes G-protein-mediated inhibition of monoamine uptake by VMAT2 expressing platelets or synaptic vesicles. In CHOVMAT2 cells G-protein-mediated inhibition of monoamine uptake can be induced by 5-hydroxytryptamine (serotonin) 1B receptor agonists, whereas ␣1 receptor agonists modulate uptake into CHOVMAT1 cells. Accordingly, 5-hydroxytryptamine 1B receptor antagonists prevent G-proteinmediated inhibition of uptake in partially filled platelets and synaptic vesicles expressing VMAT2. CHO cells expressing VMAT mutants with a shortened first vesicular loop transport monoamines. However, no or a reduced G-protein regulation of uptake can be initiated. In conclusion, vesicular content is involved in the activation of vesicle associated G-proteins via a structure sensing the luminal monoamine content. The first luminal loop of VMATs may represent a G-protein-coupled receptor that adapts vesicular filling.Communication between neurons in the central nervous system mainly occurs at specialized structures, the synapses. Variations in the input and output at synapses confer to synaptic plasticity, which involves changes at the post-and presynaptic sites, respectively. At the presynaptic site, availability and fusion competence of synaptic vesicles as well as the vesicular transmitter content contribute to the strength of postsynaptic answers.Vesicular monoamine transporters (VMATs) 2 translocate monoamines from the cytosol into the secretory vesicles of monoaminergic neurons, neuroendocrine cells, and platelets. Transport is driven by an electrochemical proton gradient (⌬H ϩ ) across the vesicular membrane, which is generated by a vacuolar H ϩ -ATPase (1). In mammals two closely related isoforms of the monoamine transporter, termed VMAT1 and VMAT2, respectively, were identified (2, 3). The transporter proteins presumably contain 12 transmembrane domains and are located on different vesicle subtypes (3-5). Both VMATs transport serotonin, dopamine, epinephrine, and norepinephrine but differ in their substrate preferences and affinities. In contrast to VMAT2, VMAT1 prefers epinephrine over norepinephrine, and the K d for serotonin uptake is around 1 M for VMAT1 but below 1 M for VMAT2. Furthermore, histamine is only transported by VMAT2. The activity of both transporters is irreversibly inhibited by reserpine, whereas tetrabenazine exclusively...

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