We attempted to identify in vitro conditions replicating those found in vivo by recording electroretinograms (ERGs) from isolated tissue. Differences in the extracellular magnesium concentration account for further quantitative differences in the kinetics of recovery from bright light flashes. We show that conditions for storing the retina can strongly affect rod sensitivity and single-photon response kinetics, and can have dramatic consequences for the health of post-receptor retinal tissue. Here we identify elements of standard recording protocols that alter the measured parameters of phototransduction. However, differences in measured rod responses indicate that the resulting quantitative picture depends strongly on recording conditions ( Doan et al., 2009 Gross and Burns, 2010). Indeed, a large body of work has made rod phototransduction the best understood G-protein cascade in biology ( Burns and Arshavsky, 2005 Burns and Pugh, 2010). This basic signaling scheme was worked out >20 years ago more recent work has focused on providing quantitative estimates of the relevant rate constants using a combination of biophysical and genetic approaches. Recovery of the resulting response depends on inactivation of these components and resynthesis of cGMP by guanylate cyclase. Activation of rhodopsin leads, through the G-protein transducin and cGMP phosphodiesterase (PDE), to a decrease in cGMP concentration, channel closure, and a decrease in current. Studies of single-photon responses offer an unprecedented opportunity to understand how activation of single GPCRs leads to the biochemical amplification that is a hallmark of G-protein signaling ( Baylor et al., 1979b Pugh and Lamb, 1993). The work described here aimed to understand the origin of these differences. These discrepancies hamper attempts to extract general principles of G-protein-coupled receptor (GPCR) function from the photoreceptors, and attempts to construct an understanding of rod vision from the molecular to the organismal level. Yet the amplitude and kinetics of the rod's responses vary by a factor of ∼2 across studies ( Calvert et al., 2000 Mendez et al., 2000 Luo and Yau, 2005 Doan et al., 2006, 2009 Krispel et al., 2006 Fu et al., 2008 Wen et al., 2009 Gross and Burns, 2010 Okawa et al., 2010). Our understanding of these processes relies on an accurate description of the rod's responses to single absorbed photons. This requires that rods detect the absorption of single photons and that the resulting signals are effectively read out by the retinal circuitry (for review, see Sharpe and Stockman, 1999 Field et al., 2005). Rod photoreceptors provide the dominant input to the visual system under conditions in which photons are scarce. The dependence of rod responses on experimental conditions will be a key consideration in efforts to extract general principles of G-protein signaling from studies of phototransduction and to relate these signals to downstream mechanisms that facilitate visual sensitivity. Several salient points emerge from these experiments: (1) recorded responses can be altered dramatically by how the retina is stored (2) the kinetics of the recovery of responses to bright but not dim flashes are strongly sensitive to the extracellular concentration of magnesium (3) experimental conditions that produce very different single-photon responses measured in single rods produce near identical derived rod responses from the electroretinogram. Third, we assayed the health of the post-receptor retinal tissue in these different conditions. Second, we compared measurements of single-cell photocurrents to estimates of rod function from in vitro electroretinograms. First, we used suction electrode recordings from single rods to compare measurements across a range of recording conditions. What accounts for these discrepancies? We used several experimental approaches to reconcile differences in published properties of rod responses. This process is one of the best understood signaling cascades in biology, yet quantitative measurements of the amplitude and kinetics of the rod's response in mice vary by a factor of ∼2 across studies.

Rod photoreceptors accomplish this task through the use of a G-protein-coupled transduction cascade that amplifies the activity of single active rhodopsin molecules. Vision in dim light, when photons are scarce, requires reliable signaling of the arrival of single photons.

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