Characteristics of Exocytosis and Endocytosis in Photoreceptors

Photoreceptors signal changes in light intensity to downstream retinal neurons through the exocytosis of glutamate-containing synaptic vesicles. The maintenance of the vesicle exocytosis and endocytosis process is essential for ongoing synaptic signaling. This study investigated the properties of exocytosis and endocytosis in photoreceptors and their role in ongoing neurotransmission. I used electrophysiology and imaging techniques to study the properties of vesicle exocytosis and endocytosis in photoreceptors. First, we examined baseline release in photoreceptors that occurs in the absence of depolarizing stimulation. We measured mEPSCs in whole cell patch clamp recordings from horizontal cells. After inhibiting Ca2+ influx and efflux and increasing intracellular Ca2+ buffering, we found that mEPSCs persisted, indicating that a portion of the baseline release occurs by a Ca2+-independent mechanism. Presynaptic recordings from rods and cones confirmed that glutamate release continues after Ca2+ is blocked. There was a decrease in frequency and amplitude of Ca2+-independent events. Visualization of individual exocytosis events by TIRF microscopy showed that Ca2+-independent release can occur at non-ribbon release sites. Following exocytosis, vesicles are retrieved by endocytosis and reenter the vesicle cycle. We measured exocytosis and endocytosis from membrane capacitance changes evoked by depolarizing steps in voltage clamped rods. Endocytosis in rods was rapid relative to other neurons with an average time constant of Ca2+ influx. Together these studies identified the sites and vesicle pools involved in Ca2+-independent baseline release from photoreceptors and found that endocytosis kinetics in rods are rapid and depend upon endocytic load and local Ca2+ levels

Lateral mobility of L-type calcium channels in synaptic terminals of retinal bipolar cells.

PURPOSE: Efficient and precise release of glutamate from retinal bipolar cells is ensured by the positioning of L-type Ca(2+) channels close to release sites at the base of the synaptic ribbon. We investigated whether Ca(2+) channels at bipolar cell ribbon synapses are fixed in position or capable of moving in the membrane. METHODS: We tracked the movements of individual L-type Ca(2+) channels in bipolar cell terminals after labeling channels with quantum dots (QDs) attached to α(2)δ(4) accessory Ca(2+) channel subunits via intermediary antibodies. RESULTS: We found that individual Ca(2+) channels moved within a confined domain of 0.13-0.15 μm(2) in bipolar cell terminals, similar to ultrastructural estimates of the surface area of the active zone beneath the ribbon. Disruption of actin expanded the confinement domain indicating that cytoskeletal interactions help to confine channels at the synapse, but the relatively large diffusion coefficients of 0.3-0.45 μm(2)/s suggest that channels are not directly anchored to actin. Unlike photoreceptor synapses, removing membrane cholesterol did not change domain size, indicating that lipid rafts are not required to confine Ca(2+) channels at bipolar cell ribbon synapses. CONCLUSIONS: The ability of Ca(2+) channels to move within the presynaptic active zone suggests that regulating channel mobility may affect release from bipolar cell terminals

Calmodulin enhances ribbon replenishment and shapes filtering of synaptic transmission by cone photoreceptors.

At the first synapse in the vertebrate visual pathway, light-evoked changes in photoreceptor membrane potential alter the rate of glutamate release onto second-order retinal neurons. This process depends on the synaptic ribbon, a specialized structure found at various sensory synapses, to provide a supply of primed vesicles for release. Calcium (Ca(2+)) accelerates the replenishment of vesicles at cone ribbon synapses, but the mechanisms underlying this acceleration and its functional implications for vision are unknown. We studied vesicle replenishment using paired whole-cell recordings of cones and postsynaptic neurons in tiger salamander retinas and found that it involves two kinetic mechanisms, the faster of which was diminished by calmodulin (CaM) inhibitors. We developed an analytical model that can be applied to both conventional and ribbon synapses and showed that vesicle resupply is limited by a simple time constant, τ = 1/(Dρδs), where D is the vesicle diffusion coefficient, δ is the vesicle diameter, ρ is the vesicle density, and s is the probability of vesicle attachment. The combination of electrophysiological measurements, modeling, and total internal reflection fluorescence microscopy of single synaptic vesicles suggested that CaM speeds replenishment by enhancing vesicle attachment to the ribbon. Using electroretinogram and whole-cell recordings of light responses, we found that enhanced replenishment improves the ability of cone synapses to signal darkness after brief flashes of light and enhances the amplitude of responses to higher-frequency stimuli. By accelerating the resupply of vesicles to the ribbon, CaM extends the temporal range of synaptic transmission, allowing cones to transmit higher-frequency visual information to downstream neurons. Thus, the ability of the visual system to encode time-varying stimuli is shaped by the dynamics of vesicle replenishment at photoreceptor synaptic ribbons

Calmodulin enhances ribbon replenishment and shapes filtering of synaptic transmission by cone photoreceptors

At the first synapse in the vertebrate visual pathway, light-evoked changes in photoreceptor membrane potential alter the rate of glutamate release onto second-order retinal neurons. This process depends on the synaptic ribbon, a specialized structure found at various sensory synapses, to provide a supply of primed vesicles for release. Calcium (Ca(2+)) accelerates the replenishment of vesicles at cone ribbon synapses, but the mechanisms underlying this acceleration and its functional implications for vision are unknown. We studied vesicle replenishment using paired whole-cell recordings of cones and postsynaptic neurons in tiger salamander retinas and found that it involves two kinetic mechanisms, the faster of which was diminished by calmodulin (CaM) inhibitors. We developed an analytical model that can be applied to both conventional and ribbon synapses and showed that vesicle resupply is limited by a simple time constant, τ = 1/(Dρδs), where D is the vesicle diffusion coefficient, δ is the vesicle diameter, ρ is the vesicle density, and s is the probability of vesicle attachment. The combination of electrophysiological measurements, modeling, and total internal reflection fluorescence microscopy of single synaptic vesicles suggested that CaM speeds replenishment by enhancing vesicle attachment to the ribbon. Using electroretinogram and whole-cell recordings of light responses, we found that enhanced replenishment improves the ability of cone synapses to signal darkness after brief flashes of light and enhances the amplitude of responses to higher-frequency stimuli. By accelerating the resupply of vesicles to the ribbon, CaM extends the temporal range of synaptic transmission, allowing cones to transmit higher-frequency visual information to downstream neurons. Thus, the ability of the visual system to encode time-varying stimuli is shaped by the dynamics of vesicle replenishment at photoreceptor synaptic ribbons