The weight of synaptic connections, which is controlled not only postsynaptically but also presynaptically, is a key determinant in neuronal network dynamics. The mechanisms controlling synaptic weight, especially on the presynaptic side, remain elusive. Using single-synapse imaging of the neurotransmitter glutamate combined with super-resolution imaging of presynaptic proteins, we identify a presynaptic mechanism for setting weight in central glutamatergic synapses. In the presynaptic terminal, Munc13-1 molecules form multiple and discrete supramolecular self-assemblies that serve as independent vesicular release sites by recruiting syntaxin-1, a soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) protein essential for synaptic vesicle exocytosis. The multiplicity of these Munc13-1 assemblies affords multiple stable states conferring presynaptic weight, potentially encoding several bits of information at individual synapses. Supramolecular assembling enables a stable synaptic weight, which confers robustness of synaptic computation on neuronal circuits and may be a general mechanism by which biological processes operate despite the presence of molecular noise.

Glutamate is the major neurotransmitter in the brain, mediating point-to-point transmission across the synaptic cleft in excitatory synapses. Using a glutamate imaging method with fluorescent indicators, we show that synaptic activity generates extrasynaptic glutamate dynamics in the vicinity of active synapses. These glutamate dynamics had magnitudes and durations sufficient to activate extrasynaptic glutamate receptors in brain slices. We also observed crosstalk between synapses-i.e., summation of glutamate released from neighboring synapses. Furthermore, we successfully observed that sensory input from the extremities induced extrasynaptic glutamate dynamics within the appropriate sensory area of the cerebral cortex in vivo. Thus, the present study clarifies the spatiotemporal features of extrasynaptic glutamate dynamics, and opens up an avenue to directly visualizing synaptic activity in live animals.synapse | spillover | fluorescence imaging | two-photon microscopy | in vivo G lutamate is the major excitatory neurotransmitter in the mammalian brain. The conventional view is that glutamate mediates synaptically confined point-to-point transmission at excitatory synapses. However, glutamate has also been suggested to escape from the synaptic cleft, generating extrasynaptic glutamate dynamics (often referred to as glutamate spillover) (1-4). Extrasynaptic glutamate dynamics has been implicated in the activation of extrasynaptic glutamate receptors via volume transmission to regulate a variety of important neural and glial functions including synaptic transmission (5, 6), synaptic plasticity (7), synaptic crosstalk (8-11), nonsynaptic neurotransmission (12, 13), neuronal survival (14), gliotransmitter release (15-17), and hemodynamic responses (18)(19)(20).Despite the immense potential physiological importance of glutamate spillover, the spatiotemporal dynamics of extrasynaptic glutamate concentration have been only inferred indirectly, and their characteristics remain elusive because of a lack of appropriate technology. Indeed, the magnitude and spatiotemporal distribution of extrasynaptic glutamate concentrations are the key determinants of physiological functions of glutamate spillover, and they are the essential factors for understanding extrasynaptic glutamate signaling. However, we have had to indirectly estimate the spatiotemporal dynamics of the glutamate spillover from its end effects mediated by glutamate receptors using electrophysiological and other means. To overcome this problem, we set out to image extrasynaptic glutamate dynamics in the brain.We developed glutamate indicators derived from the E (glutamate) optical sensor (EOS) (21). EOS is a hybrid-type fluorescent indicator consisting of the glutamate-binding domain of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunit GluR2 and a fluorescent small molecule conjugated near the glutamate-binding pocket. EOS changes its fluorescence intensity upon binding of glutamate, for which it has both high affinity and high se...

Imaging neurotransmission is expected to greatly improve our understanding of the mechanisms and regulations of synaptic transmission. Aiming at imaging glutamate, a major excitatory neurotransmitter in the CNS, we developed a novel optical glutamate probe, which consists of a ligand-binding domain of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor glutamate receptor GluR2 subunit and a small molecule fluorescent dye. We expected that such fluorescent conjugates might report the microenvironmental changes upon protein conformational changes elicited by glutamate binding. After more than 100 conjugates were tested, we finally obtained a conjugate named E (glutamate) optical sensor (EOS), which showed maximally 37% change in fluorescence intensity upon binding of glutamate with a dissociation constant of 148 nm. By immobilizing EOS on the cell surface of hippocampal neuronal culture preparations, we pursued in situ spatial mapping of synaptically released glutamate following presynaptic firing. Results showed that a single firing was sufficient to obtain high-resolution images of glutamate release, indicating the remarkable sensitivity of this technique. Furthermore, we monitored the time course of changes in presynaptic activity induced by phorbol ester and found heterogeneity in presynaptic modulation. These results indicate that EOS can be generally applicable to evaluation of presynaptic modulation and plasticity. This EOS-based glutamate imaging method is useful to address numerous fundamental issues about glutamatergic neurotransmission in the CNS.

Adenosine 5' triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, Bacillus FoF1-ATP synthase e subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo.