Presynaptic inhibition mediated by G protein-coupled receptors may involve a direct interaction between G proteins and the vesicle fusion machinery. The molecular target of this pathway is unknown. We demonstrate that Gbetagamma-mediated presynaptic inhibition in lamprey central synapses occurs downstream from voltage-gated Ca(2+) channels. Using presynaptic microinjections of botulinum toxins (BoNTs) during paired recordings, we find that cleavage of synaptobrevin in unprimed vesicles leads to an eventual exhaustion of synaptic transmission but does not prevent Gbetagamma-mediated inhibition. In contrast, cleavage of the C-terminal nine amino acids of the 25 kDa synaptosome-associated protein (SNAP-25) by BoNT A prevents Gbetagamma-mediated inhibition. Moreover, a peptide containing the region of SNAP-25 cleaved by BoNT A blocks the Gbetagamma inhibitory effect. Finally, removal of the last nine amino acids of the C-terminus of SNAP-25 weakens Gbetagamma interactions with soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. Thus, the C terminus of SNAP-25, which links synaptotagmin I to the SNARE complex, may represent a target of Gbetagamma for presynaptic inhibition.

Presynaptic inhibitory G protein-coupled receptors (GPCRs) can decrease neurotransmission by inducing interaction of G␤␥ with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. We have shown that this action of G␤␥ requires the carboxyl terminus of the 25-kDa synaptosomeassociated protein (SNAP25) and is downstream of the well known inhibition of Ca 2ϩ entry through voltage-gated calcium channels. We propose a mechanism in which G␤␥ and synaptotagmin compete for binding to the SNARE complex. Here, we characterized the G␤␥ interaction sites on syntaxin1A and SNAP25 and demonstrated an overlap of the G␤␥-and synaptotagmin I -binding regions on each member of the SNARE complex. Synaptotagmin competes in a Ca 2ϩ -sensitive manner with binding of G␤␥ to SNAP25, syntaxin1A, and the assembled SNARE complex. We predict, based on these findings, that at high intracellular Ca 2ϩ concentrations, Ca 2ϩ -synaptotagmin I can displace G␤␥ binding and the G␤␥-dependent inhibition of exocytosis can be blocked. We tested this hypothesis in giant synapses of the lamprey spinal cord, where 5-HT works via G␤␥ to inhibit neurotransmission . We showed that increased presynaptic Ca 2ϩ suppresses the 5-HT-and G␤␥-dependent inhibition of exocytosis. We suggest that this effect may be due to Ca 2ϩ

. Locomotor pattern generation is maintained by integration of the intrinsic properties of spinal central pattern generator (CPG) neurons in conjunction with synaptic activity of the neural network. In the lamprey, the spinal locomotor CPG is modulated by 5-HT. On a cellular level, 5-HT presynaptically inhibits synaptic transmission and postsynaptically inhibits a Ca 2ϩ -activated K ϩ current responsible for the slow afterhyperpolarization (sAHP) that follows action potentials in ventral horn neurons. To understand the contribution of these cellular mechanisms to the modulation of the spinal CPG, we have tested the effect of selective 5-HT analogues against fictive locomotion initiated by bath application of N-methyl-D-aspartate (NMDA). We found that the 5-HT 1D agonist, L694-247, dramatically prolongs the frequency of ventral root bursting. Furthermore, we show that L694-247 presynaptically inhibits synaptic transmission without altering postsynaptic Ca 2ϩ -activated K ϩ currents. We also confirm that 5-HT inhibits synaptic transmission at concentrations that modulate locomotion. To examine the mechanism by which selective presynaptic inhibition modulates the frequency of fictive locomotion, we performed voltage-and current-clamp recordings of CPG neurons during locomotion. Our results show that 5-HT decreases glutamatergic synaptic drive within the locomotor CPG during fictive locomotion. Thus we conclude that presynaptic inhibition of neurotransmitter release contributes to 5-HT-mediated modulation of locomotor activity.

When synaptic vesicles fuse with the plasma membrane, they may completely collapse or fuse transiently. Transiently fusing vesicles remain structurally intact and therefore have been proposed to represent a form of rapid vesicle recycling. However, the impact of a transient synaptic vesicle fusion event on neurotransmitter release, and therefore on synaptic transmission, has yet to be determined. Recently, the molecular mechanism by which a serotonergic presynaptic G-protein-coupled receptor (GPCR) regulates synaptic vesicle fusion and inhibits synaptic transmission was identified. By making paired electrophysiological recordings in the presence and absence of low-affinity antagonists, we now demonstrate that activation of this presynaptic GPCR lowers the peak synaptic cleft glutamate concentration independently of the probability of vesicle fusion. Furthermore, this change in cleft glutamate concentration differentially inhibits synaptic NMDA and AMPA receptor-mediated currents. We conclude that a presynaptic GPCR regulates the profile of glutamate in the synaptic cleft through altering the mechanism of vesicle fusion leading to qualitative as well as quantitative changes in neural signaling.

Understanding how neuromodulators regulate behavior requires investigating their effects on functional neural systems, but also their underlying cellular mechanisms. Utilizing extensively characterized lamprey motor circuits, and the unique access to reticulospinal presynaptic terminals in the intact spinal cord that initiate these behaviors, we investigated effects of presynaptic G-protein-coupled receptors on locomotion from the systems level, to the molecular control of vesicle fusion. 5-HT inhibits neurotransmitter release via a G␤␥ interaction with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that promotes kissand-run vesicle fusion. In the lamprey spinal cord, we demonstrate that, although presynaptic 5-HT receptors inhibit evoked neurotransmitter release from reticulospinal command neurons, their activation does not abolish locomotion but rather modulates locomotor rhythms. Liberation of presynaptic G␤␥ causes substantial inhibition of AMPA receptor-mediated synaptic responses but leaves NMDA receptor-mediated components of neurotransmission mostly intact. Because G␤␥ binding to the SNARE complex is displaced by Ca 2ϩ -synaptotagmin binding, 5-HT-mediated inhibition displays Ca 2ϩ sensitivity. We show that, as Ca 2ϩ accumulates presynaptically during physiological bouts of activity, 5-HT/G␤␥-mediated presynaptic inhibition is relieved, leading to a frequency-dependent increase in synaptic concentrations of glutamate. This frequency-dependent phenomenon mirrors a shift in the vesicle fusion mode and a recovery of AMPA receptor-mediated EPSCs from inhibition without a modification of NMDA receptor EPSCs. We conclude that activation of presynaptic 5-HT G-protein-coupled receptors state-dependently alters vesicle fusion properties to shift the weight of NMDA versus AMPA receptor-mediated responses at excitatory synapses. We have therefore identified a novel mechanism in which modification of vesicle fusion modes may profoundly alter locomotor behavior.