Functional maturation of the rod bipolar to AII-amacrine cell ribbon synapse in the mouse retina.

Retinal ribbon synapses undergo functional changes after eye opening that remain uncharacterized. Using light-flash stimulation and paired patch-clamp recordings, we examined the maturation of the ribbon synapse between rod bipolar cells (RBCs) and AII-amacrine cells (AII-ACs) after eye opening (postnatal day 14) in the mouse retina at near physiological temperatures. We find that light-evoked excitatory postsynaptic currents (EPSCs) in AII-ACs exhibit a slow sustained component that increases in magnitude with advancing age, whereas a fast transient component remains unchanged. Similarly, paired recordings reveal a dual-component EPSC with a slower sustained component that increases during development, even though the miniature EPSC (mEPSC) amplitude and kinetics do not change significantly. We thus propose that the readily releasable pool of vesicles from RBCs increases after eye opening, and we estimate that a short light flash can evoke the release of ∼4,000 vesicles onto a single mature AII-AC.

Bright flash response recovery of mammalian rods in vivo is rate limited by RGS9.

The temporal resolution of scotopic vision is thought to be constrained by the signaling kinetics of retinal rods, which use a highly amplified G-protein cascade to transduce absorbed photons into changes in membrane potential. Much is known about the biochemical mechanisms that determine the kinetics of rod responses ex vivo, but the rate-limiting mechanisms in vivo are unknown. Using paired flash electroretinograms with improved signal-to-noise, we have recorded the amplitude and kinetics of rod responses to a wide range of flash strengths from living mice. Bright rod responses in vivo recovered nearly twice as fast as all previous recordings, although the kinetic consequences of genetic perturbations previously studied ex vivo were qualitatively similar. In vivo, the dominant time constant of recovery from bright flashes was dramatically reduced by overexpression of the RGS9 complex, revealing G-protein deactivation to be rate limiting for recovery. However, unlike previous ex vivo recordings, dim flash responses in vivo were relatively unaffected by RGS9 overexpression, suggesting that other mechanisms, such as calcium feedback dynamics that are strongly regulated by the restricted subretinal microenvironment, act to determine rod dim flash kinetics. To assess the consequences for scotopic vision, we used a nocturnal wheel-running assay to measure the ability of wild-type and RGS9-overexpressing mice to detect dim flickering stimuli and found no improvement when rod recovery was speeded by RGS9 overexpression. These results are important for understanding retinal circuitry, in particular as modeled in the large literature that addresses the relationship between the kinetics and sensitivity of retinal responses and visual perception.

Recycling at synapses.

Synaptic vesicles in rodent neurons are recycled using at least two distinct mechanisms.

cGMP in mouse rods: the spatiotemporal dynamics underlying single photon responses.

Vertebrate vision begins when retinal photoreceptors transduce photons into electrical signals that are then relayed to other neurons in the eye, and ultimately to the brain. In rod photoreceptors, transduction of single photons is achieved by a well-understood G-protein cascade that modulates cGMP levels, and in turn, cGMP-sensitive inward current. The spatial extent and depth of the decline in cGMP during the single photon response (SPR) have been major issues in phototransduction research since the discovery that single photons elicit substantial and reproducible changes in membrane current. The spatial profile of cGMP decline during the SPR affects signal gain, and thus may contribute to reduction of trial-to-trial fluctuations in the SPR. Here we summarize the general principles of rod phototransduction, emphasizing recent advances in resolving the spatiotemporal dynamics of cGMP during the SPR.

Calcium feedback to cGMP synthesis strongly attenuates single-photon responses driven by long rhodopsin lifetimes.

Rod photoreceptors generate amplified, reproducible responses to single photons via a G protein signaling cascade. Surprisingly, genetic perturbations that dramatically alter the deactivation of the principal signal amplifier, the GPCR rhodopsin (R∗), do not much alter the amplitude of single-photon responses (SPRs). These same perturbations, when crossed into a line lacking calcium feedback regulation of cGMP synthesis, produced much larger alterations in SPR amplitudes. Analysis of SPRs from rods with and without feedback reveal that the consequences of trial-to-trial fluctuations in R∗ lifetime in normal rods are also dampened by feedback regulation of cGMP synthesis. Thus, calcium feedback trumps the mechanisms of R∗ deactivation in determining the SPR amplitude, attenuating responses arising from longer R∗ lifetimes to a greater extent than those arising from shorter ones. As a result, rod SPRs achieve a more stereotyped amplitude, a characteristic considered important for reliable transmission through the visual system.

Spatiotemporal cGMP dynamics in living mouse rods.

Signaling of single photons in rod photoreceptors decreases the concentration of the second messenger, cyclic GMP (cGMP), causing closure of cGMP-sensitive channels located in the plasma membrane. Whether the spatiotemporal profiles of the fall in cGMP are narrow and deep, or broad and shallow, has important consequences for the amplification and the fidelity of signaling. The factors that determine the cGMP profiles include the diffusion coefficient for cGMP, the spontaneous rate of cGMP hydrolysis, and the rate of cGMP synthesis, which is powerfully regulated by calcium feedback mechanisms. Here, using suction electrodes to record light-dependent changes in cGMP-activated current in living mouse rods lacking calcium feedback, we have determined the rate constant of spontaneous cGMP hydrolysis and the longitudinal cGMP diffusion coefficient. These measurements result in a fully constrained spatiotemporal model of phototransduction, which we used to determine the effect of feedback to cGMP synthesis in spatially constricting the fall of cGMP during the single-photon response of normal rods. We find that the spatiotemporal cGMP profiles during the single-photon response are optimized for maximal amplification and preservation of signal linearity, effectively operating within an axial signaling domain of ~2 µm.

Dark light, rod saturation, and the absolute and incremental sensitivity of mouse cone vision.

Visual thresholds of mice for the detection of small, brief targets were measured with a novel behavioral methodology in the dark and in the presence of adapting lights spanning ∼8 log(10) units of intensity. To help dissect the contributions of rod and cone pathways, both wild-type mice and mice lacking rod (Gnat1(-/-)) or cone (Gnat2(cpfl3)) function were studied. Overall, the visual sensitivity of mice was found to be remarkably similar to that of the human peripheral retina. Rod absolute threshold corresponded to 12-15 isomerized pigment molecules (R*) in image fields of 800 to 3000 rods. Rod "dark light" (intrinsic retinal noise in darkness) corresponded to that estimated previously from single-cell recordings, 0.012 R* s(-1) rod(-1), indicating that spontaneous thermal isomerizations are responsible. Psychophysical rod saturation was measured for the first time in a nonhuman species and found to be very similar to that of the human rod monochromat. Cone threshold corresponded to ∼5 R* cone(-1) in an image field of 280 cones. Cone dark light was equivalent to ∼5000 R* s(-1) cone(-1), consistent with primate single-cell data but 100-fold higher than predicted by recent measurements of the rate of thermal isomerization of mouse cone opsins, indicating that nonopsin sources of noise determine cone threshold. The new, fully automated behavioral method is based on the ability of mice to learn to interrupt spontaneous wheel running on the presentation of a visual cue and provides an efficient and highly reliable means of examining visual function in naturally behaving normal and mutant mice.

Control of rhodopsin's active lifetime by arrestin-1 expression in mammalian rods.

In rod photoreceptors, deactivation of the light-activated G-protein-coupled receptor rhodopsin (R*) is initiated by phosphorylation and completed through subsequent binding of visual arrestin (Arr1). The in vivo kinetics of these individual interactions have proven difficult to determine with precision since R* lifetime is much shorter than the lifetimes of downstream G-protein and effector molecules. Here, we have used a transgenic mouse line with accelerated downstream deactivation kinetics to reveal the contribution of Arr1 binding to the overall time course of rhodopsin deactivation. Photoresponses revealed that the lifetime of R* is significantly increased in rods that express half of the normal amount of Arr1, in a manner consistent with a twofold decrease in the rate of Arr1 binding across a wide range of flash strengths. A basic model of photoresponse deactivation consistent with established photoreceptor biochemistry shows that R* phosphorylation and Arr1 binding occur with a time constant of approximately 40 ms in wild-type mouse rods, much faster than previous estimates.

Enhanced arrestin facilitates recovery and protects rods lacking rhodopsin phosphorylation.

G protein-coupled receptors (GPCRs) are the largest family of signaling proteins expressed in every cell in the body and are targeted by the majority of clinically used drugs [1]. GPCR signaling, including rhodopsin-driven phototransduction, is terminated by receptor phosphorylation followed by arrestin binding [2]. Genetic defects in receptor phosphorylation and excessive signaling by overactive GPCR mutants result in a wide variety of diseases, from retinal degeneration to cancer [3-6]. Here, we tested whether arrestin1 mutants with enhanced ability to bind active unphosphorylated rhodopsin [7-10] can suppress uncontrolled signaling, bypassing receptor phosphorylation by rhodopsin kinase (RK) and replacing this two-step mechanism with a single-step deactivation in rod photoreceptors. We show that in this precisely timed signaling system with single-photon sensitivity [11], an enhanced arrestin1 mutant partially compensates for defects in rhodopsin phosphorylation, promoting photoreceptor survival, improving functional performance, and facilitating photoresponse recovery. These proof-of-principle experiments demonstrate the feasibility of functional compensation in vivo for the first time, which is a promising approach for correcting genetic defects associated with gain-of-function mutations. Successful modification of protein-protein interactions by appropriate mutations paves the way to targeted redesign of signaling pathways to achieve desired functional outcomes.