Fluorescent glutamate reporter mouse line reveals slow stochastic release from rod photoreceptor cells

The ability to see dim light in darkness requires that second-order rod bipolar cells distinguish small changes in the ongoing release of glutamate-filled vesicles from rods. This task can be facilitated by rapid release, and previous estimates of rod release rates range up to 100 vesicles/s/ribbon. To measure rates and patterns of release, we engineered mice to express a bright fluorescent glutamate reporter (iGluSnFR3.v857.GPI) in rod photoreceptor cells. After validating expression, we measured glutamate release from rods under mesopic and photopic conditions. Under mesopic conditions produced by 900-nm two-photon excitation, light flashes decreased iGluSnFR3 fluorescence consistent with diminished release. Release measured during a chirp light stimulus showed low-pass-frequency characteristics, declining in power from 1 to 10 Hz. To identify single-vesicle events, we loaded iGluSnFR3-expressing rods with dextran-conjugated pHrodo or FM4-64. Brief increases in iGluSnFR3 fluorescence due to vesicle release were accompanied by simultaneous decreases in pHrodo or FM4-64 fluorescence. Tonic release measured from changes in iGluSnFR3 fluorescence occurred stochastically at rates of 20–40 vesicles/s/ribbon under both mesopic and photopic conditions. Capacitance measurements of release from rods voltage-clamped at −40-mV rods suggested a similar rate of 38 vesicles/s/ribbon. The ability of rods to maintain similar release rates over a wide range of background intensities ensures that properties of light adaptation that arise in rods are faithfully transmitted to downstream neurons. This mouse model provides a useful tool for deciphering mechanisms by which light information is transformed at the first synapse in the retina.