Glutamate is the principal excitatory neurotransmitter, and occasionally subserves inhibitory roles, in the vertebrate nervous system. Glutamatergic synapses are dense in the vertebrate brain, at \textasciitilde1/μm3. Glutamate is released from and onto diverse components of the nervous system, including neurons, glia, and other cells. Methods for glutamate detection are critically important for understanding the function of synapses and neural circuits in normal physiology, development, and disease. Here we describe the development, optimization, and deployment of genetically encoded fluorescent glutamate indicators. We review the theoretical considerations governing glutamate sensor properties from first principles of synapse biology, microscopy, and protein structure-function relationships. We provide case studies of the state-of-the-art iGluSnFR glutamate sensor, encompassing design and optimization, mechanism of action, in vivo imaging, data analysis, and future directions. We include detailed protocols for iGluSnFR imaging in common preparations (bacteria, cell culture, and brain slices) and model organisms (worm, fly, fish, rodent).

Mechanisms that entrain and drive rhythmic epileptiform discharges remain debated. Traditionally, this quest has been focusing on interneuronal networks driven by GABAergic connections that activate synaptic or extrasynaptic receptors. However, synchronised interneuronal discharges could also trigger a transient elevation of extracellular GABA across the tissue volume, thus raising tonic GABAA receptor conductance (Gtonic) in multiple cells. Here, we use patch-clamp GABA ‘sniffer’ and optical GABA sensor to show that periodic epileptiform discharges are preceded by region-wide, rising waves of extracellular GABA. Neural network simulations that incorporate volume-transmitted GABA signals point to mechanistic principles underpinning this relationship. We validate this hypothesis using simultaneous patch-clamp recordings from multiple nerve cells, selective optogenetic stimulation of fast-spiking interneurons. Critically, we manipulate GABA uptake to suppress extracellular GABA waves but not synaptic GABAergic transmission, which shows a clear effect on rhythm generation. Our findings thus unveil a key role of extrasynaptic, volume-transmitted GABA actions in pacing regenerative rhythmic activity in brain networks.