Optogenetic Sensors

Optical imaging of light responses in the retina

Over the past 40 years, electrophysiology has been the main experimental tool for understanding how neurons in the retina respond to light and communicate with one another to process visual information. However, recent advances in optical recording methods provide new opportunities for functional studies of the retina, with several potential advantages over purely electrophysiological methods.   Optical recordings are relatively non-invasive, and depending on the type of indicator dye, can reveal different aspects of neuronal activity, including changes in intracellular ion concentrations, changes in membrane potential, and release of synaptic vesicles.   Unlike single cell electrical measurements, optical recordings can be made from many neurons at once, revealing more of the “big picture” of how the retina responds to light.

Despite these benefits, optical imaging of the retina presents a unique problem: light used to excite fluorescent indicator dyes or proteins and the resulting emitted light can inadvertently activate phototransduction in rod and cone outer segments, altering activity in their synapses and in downstream neurons. 

To overcome these limitations, we are using 2-photon microscopy with a variety of fluorescent indicator dyes to address specific questions about retinal function.  We have used small molecule dyes including the synaptic vesicle marker FM1-43 and the Ca2+ indicators fura-2 and Oregon Green BAPTA to explore different synapses in the retina of amphibians and reptiles.  In new projects, we are using genetically engineered GFP-based indicators to explore retina responses in mammalian retina.

We are particularly interested in the synapses of rod and cone photoreceptors, which transmit information to other neurons through a specialized structure called the synaptic ribbon. Unlike most synapses, ribbon synapses continuously release neurotransmitter, with the sensory stimulus (light) causing a decrease in the rate of release.  By imaging FM1-43 uptake and release with 2-photon microscopy, we have tracked the life cycle of synaptic vesicles in photoreceptor terminals (Rea et al., 2004). This process starts with endocytosis, includes interactions with the synaptic ribbon, and ends with exocytosis.   Unlike more conventional synapses, almost all of the vesicles in photoreceptor terminals are
readily available for release and contain few, if any, are held as immobilized “reserve” vesicles.  The function of the synaptic ribbon is a long-standing mystery and one of our future goals is to directly observe its interaction with synaptic vesicles to understand its role in controlling their release.

We are also examining how physiological stimuli, including light, regulate neurotransmitter release from cones (Choi et al., 2005a,b), and rods (Sheng et al., 2007).  These studies involve a combination of 2-photon microscopy to monitor dye release in the dark, and electron microscopy to quantify the number of vesicles that originally contained dye.   Because neurotransmitter release occurs through the all-or-none fusion of synaptic vesicles, the graded (“analog”) light responses of rod and cone photoreceptors must be converted into a quantal (“digital”) release event at the photoreceptor synapse.  Quantifying this “analog-to-digital” conversion is crucial for understanding the encoding of light information at this first synapse in the visual system. 

Synaptic ribbons have been thought to accelerate the delivery of vesicles to release sites at the plasma membrane, but our recent studies on cones show that the ribbon actually constrains vesicle delivery to the membrane.  Two-photon ratiometric Ca2+ imaging has given us the first view of the intra-terminal Ca2+ in light and darkness (Choi et al., 2008), essential information for understanding how vesicle release is controlled.  Electron microscopy shows that in darkness, when cones are continuously releasing neurotransmitter, the synaptic ribbon is depleted of synaptic vesicles (Jackman et al., 2009).  This indicates that vesicle resupply, not vesicle fusion, is the rate-limiting step that controls release in cones, a very surprising result.   Electrophysiological recordings of postsynaptic responses also revealed that the ribbon behaves like a capacitor, charging with vesicles in light and discharging in a phasic burst at light offset.   

This study radically changes our understanding of the role of the synaptic ribbon in signaling light responses from photoreceptors to downstream neurons in the visual system.  However, almost nothing is known of the molecular events that mediate 1) vesicle binding to the ribbon, 2) vesicle descent along the ribbon, and 3) vesicle departure from the ribbon as they approach the plasma membrane.  In ongoing experiments, we are using fluorescent probes and microscopic imaging techniques to answer these questions.  Our preliminary studies show that Rab-3a, a synaptic vesicle-associated protein, can bind to the synaptic ribbon.  We are currently testing whether this protein is necessary and sufficient for the delivery of vesicles to release sites.   If successful, these studies would provide the first detailed molecular mechanistic information of the function of the synaptic ribbon.

Two-photon imaging of synaptic vesicle release and intracellular Ca2+ is also giving us the first glimpses of other aspects of retinal signal processing, including lateral inhibition and color opponency.  These processes involve feedback inhibition of cone photoreceptors by horizontal cells in the retina.  We are using pharmacological manipulations to selectively activate or disrupt horizontal cell feedback to better understand how it modulates synaptic release from photoreceptor synapses and to explore the its underlying biophysical mechanism.   Finally, the use of activity-dependent dyes allows us to examine the behavior of 2-dimensional arrays of synapses while we project visual images on the retina.  By visualizing many neurons simultaneously, optical recording methods provide a unique opportunity to directly see how retinal information is processed at sequential synaptic layers.