In retinitis pigmentosa (RP), primary rod degeneration leads to the secondary degeneration of cones, resulting in loss of normal retinal input. Retinal function is further disrupted by downstream retinal remodeling. The mechanisms underlying these degenerative processes are currently incompletely understood. In my thesis research, I use whole-cell electrophysiology recordings from retinal slices to investigate changes in retinal physiology during retinal degeneration.

In the first section of my thesis, I investigate the changes in the rod-to-rod bipolar cell synapse during the early stages of degeneration in the CNGB1 KO mouse model. Previous studies show that rod bipolar cells lack light-evoked responses prior to the loss of light responses in rods, despite intact rod-to-rod bipolar cell synapses. Using a weak mGluR6 antagonist, CPPG, I show that although rod bipolar cells lose light-evoked responses they retain the molecular machinery required to support mGluR6-driven responses. These results indicate that the breakdown in synaptic transmission between rods and rod bipolar cells is most likely due to presynaptic dysfunction with impaired glutamate release from rods.

In the second section of my thesis I characterized changes in cone membrane physiology during secondary cone degeneration by performing whole-cell patch clamp recordings from degenerating cones in rd10 mice. I show that, despite significant morphological changes, including the loss of the outer segment and cone pedicle, cones maintain relatively normal membrane physiology. What’s more, cones continue to produce small light-evoked responses even at late stages of degeneration, and these responses are transmitted on to second-order neurons. These findings are encouraging for future research aimed at reactivating dormant cones to restore vision to patients with RP.

In the final section of my thesis I explore the evolutionary origins of one of the vertebrate retina’s most unique features: the use of the metabotropic glutamate receptor in the ON bipolar cell pathway. My results indicate that the division of light information into ON and OFF pathways at the first synaptic connection and the mediation of the ON pathway through a metabotropic glutamate receptor evolved over 500 million years ago and is a fundamental feature of the vertebrate retina.

Vision begins in the retina, where photoreceptors have the task of discriminating incomingphotons from intrinsic cellular noise. Vertebrates have evolved a duplex retina, in which rod and cone photoreceptors respond over a broad range of light intensities from single photons to bright light. Rods evolved from less sensitive cone-like photoreceptors and are responsible for mediating the lowest 8 orders of magnitude of light intensity. At the lowest threshold of vision, rods detect single photons and transmit signals to downstream neurons in a specialized circuit. In the rod visual pathway, tens of rods converge onto a single rod bipolar cell, where noise from the rods is filtered, and rod signals are processed for further downstream transmission. It is unclear which evolutionary changes imparted such sensitivity to rods, and how these advantages allow for rods to discriminate single photons from intrinsic noise. In downstream bipolar cells, the mechanism for setting the filtering threshold is unknown. Further, it is unclear what implications of the thresholding of rod bipolar cells have on vision in scotopic light levels. In this thesis, I investigated the mechanisms that work together to define the threshold of vision. To gain understanding of the nature of vision at the absolute threshold, I worked with three main goals in mind: • Goal 1: Determine the set of changes that make feasible the incredible sensitivity of rods. • Goal 2: Assess contributions of transduction proteins to the detection of single photons versus intrinsic noise. • Goal 3: Measure the ability of rod bipolar cells to shape and parse rod signals over several orders of magnitude of ambient light levels. To achieve these goals, I collected single-cell electrophysiological from mouse rods and rod bipolar cells. I analyzed the data with statistical models to determine parameters that described the mechanisms of visual threshold and adaptation. I also used statistical modeling to help validate results of mathematical modeling from our collaborators in the Reingruber Lab (Paris, France), which show that the important differences between rods and cones are reflected in the cone as a reduced outer segment volume, a decreased amplification through the transduction cascade, a faster turnover rate of the cyclic guanosine monophosphate second messenger, and an accelerated rate of decay of the phosphodiesterase (PDE) enzyme (goal 1). Through analysis of intrinsic noise in rods, I showed that PDE is the critical transduction element both setting the noise level and limiting the detection threshold and variability for single-photon absorptions (goal 2). Further, measurements in rod bipolar cells revealed that rod bipolar cells have a calcium-dependent feedback mechanism that allows rod bipolar cells to respond robustly to single-photon absorptions in the minority of the rods, while continuing to signal in brighter backgrounds that also cause rod adaptation (goal 3). Taken together, my work has helped delineate the fundamental mechanisms of signal detection and processing at the threshold of vision and over the dynamic range of rod vision.

Structural studies on the cGMP-specific phosphodiesterase (PDE6) found in vertebrate rod photoreceptors (PDE6) reveal that in addition to its two catalytic binding sites, PDE6 possesses two noncatalytic sites that also bind cGMP. These sites are thought potentially to influence the mechanism through which vertebrate photoreceptors adapt to light. By assessing the electrophysiological response via suction-electrode recordings of transgenic mice (NCBS-1) which have a knockout of one of the two noncatalytic sites, we determined whether there were changes in the physiological amplitude and time course of the photoresponse. We are able to characterize the presence of an altered phenotype in the NCBS-1 rod light response, wherein the waveform is lengthened and responses for similar intensities of light are larger than those in the wild-type mouse. These results confirm a modest alteration of the adaptation mechanism but show that the non-catalytic sites in some way control expression or trafficking of the PDE6.