Optical analysis of the action range of glutamate in the neuropil

The wiring scheme of neurons is key to the function of the brain. Neurons are structurally wired by synapses and it is a long-held view that most synapses in the CNS are sufficiently isolated to avoid cross-talk to AMPA receptors of neighboring synapses. Here we report in hippocampal brain slices that quantal glutamate release activated optical reporter proteins >1.5 μm distant to the releasing synapse. 2P-glutamate uncaging was used to quantitatively probe glutamate spread in the neuropil. Releasing ~35000 molecules of glutamate (~5 vesicles) at a distance of 500 nm to a spine generated an uncaging EPSC reaching ~30% of the quantal amplitude at synaptic AMPA-Rs. The same stimulus activated ~70% of the quantal amplitude at NMDA-Rs and still generated clear current and calcium responses when applied at >= 2 μm remote to the spine. Extracellular spread of glutamate on the sub-micrometer scale appeared cooperative and caused supra-additive activation of AMPA-Rs in a spine. These observations are not predicted by previously used models of glutamate diffusion in the neuropil. An extracellular glutamate scavenger system weakly reduced field potential responses but not the quantal amplitude, indicating that a cross-talk component regularly contributes to synaptic transmission. Our data suggest that slight synaptic crosstalk responses at AMPA receptors of ~2-4 adjacent synapses may be common (>70 synapses for NMDA receptors). Such broadcasting of synaptic signals to very local neighborhoods could stabilize network learning performance and allow for integration of synaptic activity within the extracellular space.