The discovery of the ability of rod photoreceptors to signal single photons

The Journal of General Physiology has a rich tradition of publication in sensory physiology, particularly in vision science. This includes much research on rod and cone visual pigments and their photochemistry, on visual retinoid physiology, and on phototransduction. The Journal continues to attract such research, not only because of this history, but also because of its transparent reviewing principles and its outstanding production quality. Cited 1,195 times according to Google Scholar, the paper on the absolute sensitivity of human vision that discovered the ability of rod photoreceptors to respond to single photons (Hecht et al., 1942) stands out for its importance and enduring impact.

A cardinal principle of quantum mechanics is that all exchange of energy between the electromagnetic field and matter takes place in discrete packets or quanta. This principle had been firmly established by 40 years of physics and spectroscopic chemistry when Hecht et al. made their meticulous measurements of the threshold energy for detection of a small, brief 500 nm visual target by dark-adapted human observers¹. Reported as the energy at the 60th percentile of ogival frequency of seeing curves (Fig. 1 A), the thresholds measured at the cornea for their four primary observers ranged from 2.1 to 5.7 × 10⁻¹⁷ J, equivalent to 48–148 photons according to Planck’s formula (Table 1).

Hecht et al. drew several enduring mechanistic conclusions. First, as there were inevitable losses in light transmission through the ocular media, the number of photons captured by rhodopsin² in rods was less than the number incident on the cornea by a factor of 2–10 or more. Second, because visual threshold required the capture of only a small number of photons, Poisson statistics implied substantial fluctuations in the number captured trial-by-trial from each nominal energy flash. Third, because the region of the retina upon which the target was imaged subtended several hundred rod photoreceptors, it was highly improbable that any individual rod captured more than one photon at threshold. Thus, Hecht et al. incontrovertibly drew a profound conclusion that has stood the test of time, namely that rod photoreceptors had attained the limit of light sensitivity dictated by quantum physics. In other words, rods were capable of generating single-photon responses (SPRs) that propagated through the retina and created reliable signals in the central nervous system (CNS).

The impact of Hecht et al.’s investigation on sensory physiology has been extensive and includes inspiring numerous investigations into aspects of the molecular mechanisms of phototransduction, guiding research into the retinal circuitry underlying night vision, and providing novel insight into mechanisms that cope with (and even exploit) noise in neuronal circuits. These topics will be reviewed in this paper after a brief summary of subsequent experiments that confirmed and extended the findings of Hecht et al. (1942).

The findings of Hecht et al. (1942) have been replicated in several human psychophysical studies and in a recent behavioral study of mice (Fig. 1 and Table 1). Several factors contribute to variation in threshold between experiments, including the target size, duration, and retinal location; optima for these factors reflect variation in rod density and in properties of the underlying retinal circuitry such as receptive field size, which vary systematically with eccentricity in humans. Another critical factor affecting threshold is the observer’s criterion for reporting a target as seen (i.e., giving a “yes” response) on any given trial. Hecht et al. (1942) hypothesized that the Poisson fluctuations in photon capture were the primary source of variation underlying the trial-by-trial variability in detection of targets of the same nominal energy and that differences among their observers arose from the adoption of different criteria for the strength of the sensation required for a yes response. However, as developed initially in vision research by Barlow (1956) and further by him and many others, threshold detection is best understood as a signal/noise discrimination (Green and Swets, 1966), with noise arising from intrinsic retinal processes (Barlow et al., 1957). In the signal detection framework, systematic manipulation of the criterion (e.g., with confidence-rating scales) revealed that observers could indeed reliably detect stimuli whose average energy at the cornea was threefold or more lower than the average 90 photons of Hecht et al.’s experiments (Sakitt, 1972; Teich et al., 1982; Tinsley et al., 2016). Strong support for the validity of this framework arose from the “receiver operating characteristic” relationship between detection probability and false positive rate (Green and Swets, 1966); observers could detect lower energy stimuli with high probability on stimulus-null trials only at the cost of increased false positive responses, which were reasonably presumed to be triggered by retinal noise. In this context, Hecht et al.’s observers were understood to have adopted high criteria to avoid making false positive responses to very dim flashes that they hadn’t “really” seen. Models of detection that incorporate intrinsic neural noise naturally explained the relationship between detection and false positive rates at low stimulus energies (Sakitt, 1972; Teich and Prucnal, 1977; Field et al., 2005; Naarendorp et al., 2010; Koenig and Hofer, 2011). In such models, an event counter serves as a detector that reaches the detect state when the count on a trial exceeds a criterion number as postulated by Hecht et al. However, spontaneous noise events not generated by the stimulus contribute to each count. Indeed, as originally hypothesized by Barlow et al. (1957) and subsequently confirmed (below), rhodopsin, the marvelous molecule that can signal the capture of one photon, can also spontaneously trigger photon-like events in the absence of light. In summary, although confirming the fundamental findings of Hecht et al. (1942) and strongly affirming their conclusion that rods are able to generate SPRs, subsequent experiments and analyses revealed that intrinsic retinal noise, as well as Poisson fluctuations in the number of captured photons, was an important determinant of absolute visual sensitivity.

A recent human threshold study used an optical technology that enabled identification of trials in which exactly one photon was delivered to the eye (Tinsley et al., 2016). In their two-alternative forced choice (2AFC) detection task, subjects in one-photon trials made the correct choice 51.6% of the time, which was a success rate 1.6% higher than the 50% chance performance level, yet statistically reliable (P = 0.05, 2,420 one-photon trials). These and other results in the study make the case that a well-trained observer in a task that pushes their criterion to its lowest level can, albeit infrequently, correctly discriminate trials producing a single SPR from trials with noise alone (Tinsley et al., 2016). Although reaching the ultimate physically allowable limit of visual system performance, these results are not incommensurate with prior studies (Table 1). For example, the mouse whose data are shown in Fig. 1 A was successful 8% of the time on trials when 12 photons on average were delivered to its eye, a success rate reliably greater than its false alarm rate, 1.0% (130 stimulus trials, 500 blank trials; P < 10⁻⁴), and the average number of SPRs in nominal 12-photon trials was likely one third or less than the number of photons arriving at the cornea. A broad issue raised by one-photon detection trials is whether the CNS can extract information useful to the organism from a stimulus that can only be detected a small fraction of the time it is available. The conventional definition of threshold as the energy corresponding to the midpoint of the frequency of seeing curve corresponds to an ∼50% false negative rate and, to some extent, embodies the hypothesis that stimuli that give rise to much higher false negative rates may not have much value to an organism. It is notable that the subjects of the Tinsley et al. (2016) study, even in the performance-enhancing 2AFC task, had thresholds comparable to those of subjects in the other studies (Table 1). For an organism in a more natural context, both false negatives (type II errors) and false positives (type I errors) will usually have a cost. In this context, it is notable that mice, like the subjects of Hecht et al.’s study, naturally maintained a low false positive rate of 1–2% (Naarendorp et al., 2010) and thus implicitly maintained a high criterion.

Electrical recordings of SPRs from vertebrate photoreceptors were made for the first time by Baylor, Lamb, and Yau from single toad rods with suction electrodes (Baylor et al., 1979b; Yau et al., 1977). In a companion study, the authors characterized the rod circulating (dark) current and the intensity dependence and kinetics of its suppression by light (Baylor et al., 1979a), confirming the discovery by Hagins et al. (1970) in rat retinal slices of the light-suppressible, outer segment–inward, inner segment–outward dark current. The critical evidence for SPRs was that for appropriately weak flash strengths, the probability of a response followed Poisson statistics with the frequencies of trials with no events and of trials with events appropriately dependent on flash strength Baylor et al., 1979b. Subsequent work characterized the SPRs of primate rods (Baylor et al., 1984), followed by numerous studies of SPRs in mouse rods (e.g., Rieke and Baylor, 1998; Field et al., 2005; Gross et al., 2015). One key finding of all these studies was that a single rhodopsin isomerization generated an SPR that suppressed 2–5% of the dark current at the response peak. Because the inward current was found to be uniformly distributed along the outer segment (Baylor et al., 1979a), such suppression implied the axial spread of a signal along the outer segment from the disc membrane where the isomerized rhodopsin (R*) resided and thus further implied the existence of an internal messenger. A second key finding was that SPRs were highly reproducible in shape and amplitude, with a coefficient of variation (SD/mean) of amplitude of 0.2 (amphibian rods) to 0.3 (mammalian rods). A third fundamental finding of the suction electrode studies was that in darkness, rods generated spontaneous events whose amplitude and kinetics were indistinguishable from the light-stimulated SPRs. These spontaneous photon-like events, which were naturally attributed to thermally triggered activation of rhodopsin, were relatively infrequent: 0.02 s⁻¹ in toad rods at room temperature (Baylor et al., 1980), 0.006 s⁻¹ in primate rods (Baylor et al., 1980), and 0.012 s⁻¹ in mouse rods (Burns et al., 2002) at body temperature. Because toad rods contain 2 × 10⁹ rhodopsin molecules (mammalian rods, ∼10⁸), these dark events revealed that the rhodopsin molecule, today understood to be a GPCR covalently bound to its natural “inverse ligand,” 11-cis-retinal, is extraordinarily stable; the thermal activation rate at room temperature corresponds to a rate constant of once per 50,000 years. Nonetheless, for their rarity in individual rods, because of the very high convergence of signaling in successive layers of the retina, these spontaneous events create “dark light” that sets a limit on the sensitivity of the dark-adapted eye and the visual system as a whole, as further discussed below.

Phototransduction is the molecular and cellular process by which the isomerization of the covalently liganded 11-cis-retinal chromophore of an opsin GPCR leads to the production of an electrical response in a rod or cone photoreceptor cell. Grounded on the principle that isomerization of the chromophore is the initial event in rod-mediated vision (Wald and Brown, 1958; Wald et al., 1963), at the time when SPRs were first reported, research into the molecular mechanisms of phototransduction was advancing rapidly, culminating in the discoveries of amplified G protein and cGMP phosphodiesterase activation and of the CNG-gated channels of the plasma membrane (Yee and Liebman, 1978; Fung et al., 1981; Fesenko et al., 1985; Stryer, 1986). The electrical recording of SPRs, however, provided much inspiration and a clear focus for mechanistic investigations. For example, the hypothesis that a diffusible internal messenger carried the signal between rod disc membranes and the plasma membrane received strong support from the fact that an SPR involved the closure of channels at micrometer distances along the outer segment from the disc membrane locus of the R* (Baylor et al., 1979b; Lamb et al., 1981; Pugh and Cobbs, 1986). Similarly, the realization that a single R* could lead to suppression of a few percent of the outer segment membrane current implied that the biochemical mechanism was strongly amplifying, so that the theory of phototransduction had to account for this amplification (Lamb and Pugh, 1992; Pugh and Lamb, 1993; Leskov et al., 2000). Another key aspect of the SPR that inspired research into molecular mechanisms was its reproducibility; how could the activity time course of a single GPCR give rise to so stereotypic an electrical response when one-step stochastic deactivation of R* predicts a coefficient of variation of unity? Although some debate continues on these issues, it now seems clear that SPR reproducibility is achieved primarily by three mechanisms: (1) high gain of the fully active R*, such that it initially activates G proteins at the rate of hundreds per second; (2) timed deactivation of R* by multiple phosphorylation, leading to gradually increasing affinity of the positively charged binding surface of the capping protein arrestin for the increasingly phosphorylated state; and (3) feedback activation of guanylate cyclase, whereby the decline in Ca²⁺_i that accompanies the closure of CNG channels dampens the variability that would otherwise result from stochastically longer R* lifetimes (Gross et al., 2012).

A profound implication of the experiments of Hecht et al. (1942) and its replications and extensions is that a small number of rod SPRs can generate a signal that propagates reliably through the retina and into the CNS, where the ultimate correlates of detection and behavioral response generation must occur. There are two related aspects to this implication: (1) the cellular and subcellular anatomy of the neural circuitry of the post-rod SPR signals and (2) the signal processing in that circuitry. These topics have been the focus of a great body of research (Tsukamoto et al., 2001; Field et al., 2005; Field and Sampath, 2017). In regard to the anatomy underlying SPR signaling, research has revealed that evolution fashioned a unique retinal circuit to carry the SPR signal in the most dark-adapted state, namely via rod bipolar cells (RBCs)→AII amacrine cells→ON-α/β retinal ganglion cells (RGCs; Tsukamoto et al., 2001; Takeshita et al., 2017). This “starlight” pathway is highly convergent, such that thousands of rods signal to a single ON-β or ON-α RGC, and clearly operates over much of the intensity regime of night vision (Fig. 1 B). This circuit also appears grafted into more primitive versions of the retina, affording an interesting case in which the evolution of a multicellular neuronal circuit can be related to a specific function: SPR signaling (Lamb et al., 2007). In regard to the processing, both the overall architecture of the SPR-signaling circuits and their subcellular elements have been found to have properties that aid in noise reduction and signal transmission efficiency. For example, the rod→RBC synapse has a nonlinearity that serves to minimize transmission of non-SPR rod membrane noise (Field and Rieke, 2002; Berntson et al., 2004). Other functional aspects of the circuitry, such as coincidence detection mechanisms at the RGC level, remain under intense investigation (Ala-Laurila and Rieke, 2014) and are contributing novel mechanistic insight into how intrinsic noise is processed by neuronal circuits to improve signal detection.

This admittedly inadequate paper has attempted to briefly summarize a large body of scientific investigations, ranging from characterization of the molecular events initiated by a single captured photon to signal transmission in the visual systems of humans and mice, that can trace their lineage back to the seminal work of Hecht et al. (1942). (For other recent reviews of this topic, see Nelson [2016] and Field and Sampath [2017].) The deduced and subsequently directly measured ability of a single photoisomerized rhodopsin molecule to generate a reliable signal that propagates through the visual system is an extraordinary feat of evolution. This discovery will no doubt continue to inspire novel investigations into the rich array of mechanisms that make it possible, from the extremely stable nature of the inverse-liganded rhodopsin GPCR and its photoactivation into a form that triggers a high-gain and highly reproducible SPR, to signal transmission mechanisms in the retina and visual pathways that filter noise and amplify rod SPR signals. Perhaps two examples of extant questions arising from this heritage can serve to amplify this point.

In their work characterizing the spontaneous activation of rhodopsin, Baylor et al. (1980) reported that the activation energy (22 kcal/mol) and activation entropy (−35 e.u.) of the spontaneous photon-like event-generating process were essentially the same as those for 11-cis-retinal in detergent (Hubbard, 1966), observations that greatly strengthen the conclusion that thermal isomerization of the rhodopsin chromophore underlies these SPR-like events. However, the rate constant of isomerization of the chromophore in digitonin was ∼1,000-fold higher than that derived from the spontaneous event rate of rods. Given the strong evidence that the spontaneous events indeed arose from chromophore isomerization, collectively, the results imply that the rhodopsin-binding pocket greatly stabilizes the 11-cis-retinal chromophore. Indeed, in the absence of this stabilization, the rate of spontaneous events in a typical mammalian rod would be at least 6 s⁻¹, a rate that would swamp the ultrasensitive rod bipolar cells whose signaling current is half-maximal when, on average, each of the 20–50 rods in its receptive field captures one photon. Clearly, the physicochemical mechanisms of stabilization have been of profound importance for vision, for without this stabilization, night vision would literally be blinded by a blizzard of rod-generated dark light. It remains a challenge for the future to understand the mechanisms of chromophore stabilization by the protein rhodopsin, for example, by reducing some of the vibrational modes of the chromophore (Kim et al., 2003; Ala-Laurila et al., 2004). This effort should be abetted by the rich universe of opsin GPCRs, which vary not only in their spectral tuning but also in their chromophore stability (Ala-Laurila et al., 2004).

The insight that evolution fashioned a highly convergent neuronal circuit (RBC→AII amacrine→ON-RGC) specifically tailored for signaling SPRs raises many challenging questions for developmental and evolutionary biology. Because the circuit has such a clear and distinct function, it may serve as a textbook case of the coevolution of molecular, cellular, and transcellular features that serve a well-defined physiological function: high-fidelity transmission of SPR events in a noisy background. Biophysically accurate modeling of the circuits may continue to identify novel molecular mechanisms at various scales that contribute to the remarkable signal/noise ratio that has been achieved under selection pressure for vision to operate in photon-starved environments, and thus for SPR signal transmission from rods to the CNS.

Olaf S. Andersen served as editor.