Measuring light -regulated synaptic activity in the intact vertebrate retina using optical imaging methods

The retinal photoreceptors are the primary sensors that detect light and transmit light information to the rest of the visual system. Here we use optical imaging techniques to elucidate how changes in tonic release rate from the photoreceptor synapse encode and transmit light information to downstream neurons. We label retinal synapses with the fluorescent activity-dependent dye, FM 1-43, and visualize release using two-photon microscopy, which uses infrared pulses of light and can image deep in the intact retina without interfering with photoreceptor function. This combination of optical tools enables not only measurement of light-dependent activity of an array of retinal synapses within intact local networks in the vertebrate retina, but also provides single cell and subcellular spatial resolution. We utilize the all-cone retina of anole lizard to directly measure the tonic release from photoreceptor synapse. Using time-lapse fluorescence microscopy and electron microscopy, we find that at rest in darkness, the cone synapse releases ∼250 vesicles/s. The release rate decreases linearly with light intensity and become almost completely suppressed to 12 vesicles/s in saturating light. Thus, the cone compresses light information over 4 to 5 log units of intensity it detects in the outer segment into a ∼20-fold change in synaptic release. Since calcium influx directly regulates release, we then use fluorescent calcium indicator dyes to examine light-dependent changes in calcium level within the cone synaptic terminal. We find that the resting calcium level is 100-250 nM in darkness and reduces to 35-50 nM in saturating light. Dimming the light intensity 10-fold reduces calcium level by half, suggesting synaptic calcium concentration is not linearly related to intensity. Analysis of high resolution spatial profiles of the cone synapse shows that light-regulated change in calcium level is accentuated near the synaptic ribbons where voltage-gated calcium channels are thought to be localized. Putting together the optical measurements of tonic release and calcium dynamics, we find calcium vs. release is a linear relation in the cone photoreceptor synapse.