The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit saturating activation kinetics and are excluded from post-synaptic densities, limiting their ability to distinguish synaptic from extrasynaptic glutamate. Using a multi-assay screen in bacteria, soluble protein, and cultured neurons, we generated novel variants with improved kinetics and signal-to-noise ratios. We also developed surface display constructs that improve iGluSnFR’s nanoscopic localization to post-synapses. The resulting indicator, iGluSnFR3, exhibits rapid non-saturating activation kinetics and reports synaptic glutamate release with improved linearity and increased specificity versus extrasynaptic signals in cultured neurons. In mouse visual cortex, imaging of iGluSnFR3 at individual boutons reported single electrophysiologically-observed action potentials with high specificity versus non-synaptic transients. In vibrissal sensory cortex Layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit low in vivo signal-to-noise ratios, saturating activation kinetics and exclusion from postsynaptic densities. Using a multiassay screen in bacteria, soluble protein and cultured neurons, we generated variants with improved signal-to-noise ratios and kinetics. We developed surface display constructs that improve iGluSnFR’s nanoscopic localization to postsynapses. The resulting indicator iGluSnFR3 exhibits rapid nonsaturating activation kinetics and reports synaptic glutamate release with decreased saturation and increased specificity versus extrasynaptic signals in cultured neurons. Simultaneous imaging and electrophysiology at individual boutons in mouse visual cortex showed that iGluSnFR3 transients report single action potentials with high specificity. In vibrissal sensory cortex layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

Visual information in birds is to great extent processed in the optic tectum (TeO), a prominent laminated midbrain structure. Retinal input enters the TeO in its superficial layers, while output is limited to intermediate and deeper layers. In addition to visual information, the TeO receives multimodal input from the auditory and somatosensory pathway. The TeO gives rise to a major ascending tectofugal projection where neurons of tectal layer 13 project to the thalamic nucleus rotundus, which then projects to the entopallium. A second tectofugal projection system, called the accessory pathway, has however not been studied as thoroughly. Again, cells of tectal layer 13 form an ascending projection that targets a nucleus known as either the caudal part of the nucleus dorsolateralis posterior of the thalamus (DLPc) or nucleus uveaformis (Uva). This nucleus is known for multimodal integration and receives additional input from the lateral pontine nucleus (PL), which in turn receives projections from layer 8–15 of the TeO. Here, we studied a particular cell type afferent to the PL that consists of radially oriented neurons in layer 9. We characterized these neurons with respect to their anatomy, their retinal input, and the modulation of retinal input by local circuits. We found that comparable to other radial neurons in the tectum, cells of layer 9 have columnar dendritic fields and reach up to layer 2. Sholl analysis demonstrated that dendritic arborization concentrates on retinorecipient layers 2 and 4, with additional arborization in layers 9 and 10. All neurons recorded in layer 9 received retinal input via glutamatergic synapses. We analyzed the influence of modulatory circuits of the TeO by application of antagonists to γ-aminobutyric acid (GABA) and acetylcholine (ACh). Our data show that the neurons of layer 9 are integrated in a network under strong GABAergic inhibition, which is controlled by local cholinergic activation. Output to the PL and to the accessory tectofugal pathway thus appears to be under strict control of local tectal networks, the relevance of which for multimodal integration is discussed.