ABSTRACT Extracellular pH impacts on neuronal activity, which is in turn an important determinant of extracellular H + concentration. The aim of this study was to describe the spatio-temporal dynamics of extracellular pH at synaptic sites during neuronal hyperexcitability. To address this issue we created ex.E 2 GFP, a membrane-targeted extracellular ratiometric pH indicator that is exquisitely sensitive to acidic shifts. By monitoring ex.E 2 GFP fluorescence in real time in primary cortical neurons, we were able to quantify pH fluctuations during network hyperexcitability induced by convulsant drugs or high-frequency electrical stimulation. Sustained hyperactivity caused a pH decrease that was reversible upon silencing of neuronal activity and located at active synapses. This acidic shift was not attributable to the outflow of synaptic vesicle H + into the cleft nor to the activity of membrane-exposed H + V-ATPase, but rather to the activity of the Na + /H + -exchanger. Our data demonstrate that extracellular synaptic pH shifts take place during epileptic-like activity of neural cultures, emphasizing the strict links existing between synaptic activity and synaptic pH. This evidence may contribute to the understanding of the physio-pathological mechanisms associated with hyperexcitability in the epileptic brain.

Synapsins are synaptic vesicle (SV)-associated phosphoproteins involved in the regulation of neurotransmitter release. Synapsins reversibly tether SVs to the cytoskeleton and their phosphorylation by serine/threonine kinases increases SV availability for exocytosis by impairing their association with SVs and/or actin. We recently showed that synapsin I, through SH3- or SH2-mediated interactions, activates Src and is phosphorylated by the same kinase at Tyr301. Here, we demonstrate that, in contrast to serine phosphorylation, Src-mediated tyrosine phosphorylation of synapsin I increases its binding to SVs and actin, and increases the formation of synapsin dimers, which are both potentially involved in SV clustering. Synapsin I phosphorylation by Src affected SV dynamics and was physiologically regulated in brain slices in response to depolarization. Expression of the non-phosphorylatable (Y301F) synapsin I mutant in synapsin-I-knockout neurons increased the sizes of the readily releasable and recycling pools of SVs with respect to the wild-type form, which is consistent with an increased availability of recycled SVs for exocytosis. The data provide a mechanism for the effects of Src on SV trafficking and indicate that tyrosine phosphorylation of synapsins, unlike serine phosphorylation, stimulates the reclustering of recycled SVs and their recruitment to the reserve pool.

Synapsins are synaptic-vesicle-associated phosphoproteins implicated in the regulation of neurotransmitter release and excitability of neuronal networks. Mutation of synapsin genes in mouse and human causes epilepsy. To understand the role of the highly conserved synapsin domain E in the dynamics of release from mammalian inhibitory neurons, we generated mice that selectively overexpress the most conserved part of this domain in cerebellar Purkinje cells. At Purkinje-cell–nuclear-neuron synapses, transgenic mice were more resistant to depression induced by short or prolonged high-frequency stimulations. The increased synaptic performance was accompanied by accelerated release kinetics and shorter synaptic delay. Despite a marked decrease in the total number of synaptic vesicles, vesicles at the active zone were preserved or slightly increased. The data indicate that synapsin domain E increases synaptic efficiency by accelerating both the kinetics of exocytosis and the rate of synaptic vesicle cycling and decreasing depression at the inhibitory Purkinje-cell–nuclear-neuron synapse. These effects may increase the sensitivity of postsynaptic neurons to inhibition and thereby contribute to the inhibitory control of network activity.