Retinal rods and cones are highly specialized neurons that transform light into an electrical signal (see Chapter 18 ) and provide the sensory input for vision. In contrast to most neurons, rods and cones maintain a relatively depolarized membrane potential at rest (in darkness), and when stimulated (by light) they decrease Na + entry by closing ion channels. The resultant hyperpolarization closes Ca 2+ channels at the synapse, and the ensuing fall in intracellular Ca 2+ reduces an ongoing vesicular release of the neurotransmitter glutamate onto second-order neurons. This chapter describes the signaling properties of rods and cones that subserve vision in dim and bright light, respectively. An overview of the voltage changes induced by light will be followed by a description of the properties of the photocurrent and then an explanation of how voltage-gated inner segment conductances shape the final voltage response. For readers interested in the underlying molecular designs and mechanisms, summaries on ion channels and exchangers are included. Although the retinal photoreceptors of all vertebrates operate similarly, there are some qualitative, as well as quantitative, differences. Therefore, wherever possible, we will focus on primate photoreceptors.
Photovoltage response to flashes
In darkness, rods and cones maintain a membrane potential near –40 mV. When stimulated by flashes, rods and cones do not “fire” action potentials but instead respond with slow graded hyperpolarizations ( Fig. 19.1 ) that in some cells exceed 25 mV (e.g., ). The hyperpolarization then spreads passively to the synapse. In comparing rod and cone responses, the latter are considerably faster and require many more photons. Photoisomerization of 75 rhodopsins gives rise to a half-maximal response in rods, whereas in cones it takes close to 1000 photoisomerizations (estimated from , ).
The amplitude of the single photon response in rods is about a millivolt, a few percent of the maximal response. , , The time to a peak of approximately 200 ms is relatively slow, considering that Olympic sprinter Usain Bolt covered more than two meters in the same time span. The recovery to baseline is even slower and may take over a second for completion. The slow time course of the single photon response provided compelling evidence for an internal second messenger(s) and for amplification steps in the signaling pathway. The duration can be specified as an integration time, calculated by dividing the response integral by the peak amplitude. For rods, the integration time of the quantal response is several hundred milliseconds. Although cones are less sensitive than rods, they too “respond” to single photons, but the amplitude of their quantal response is much smaller and does not emerge from the baseline noise. The cone response is faster, with a time to peak of 30 ms and integration time of only 25 ms. These values are approximate because responses of foveal cones are slower than those in the periphery, and responses of blue cones are slightly slower than those of red and green cones. , Response kinetics and sensitivity are intimately linked, and the relationship is discussed further in the chapter. The waveforms of rod and cone flash responses change with flash strength. Responses peak sooner and the initial recovery is faster as the flash strength increases, but with responses greater than half maximal, recovery stalls to a plateau before the final recovery. In cones, the recovery overshoots the baseline before the final recovery, whereas in rods, the plateau slowly returns to baseline without an overshoot.
Background light attenuates the response to a flash as the cells adapt (for discussions of the underlying mechanisms, see Chapters 18 and 20 ). The dim flash response reduces in size twofold during exposure for several tens of seconds to backgrounds producing approximately 150 photoisomerizations per second in rods and approximately 8700 photoisomerizations per second in cones. Background light quickens flash responses in rods but has little effect on response kinetics in cones (ignoring the rod component of the response in cones communicated electrotonically, see Chapter 22 ).
Photocurrent response to flashes
The photon response derives from a decrease in the number of open cyclic nucleotide-gated (CNG) channels that changes the flow of ions across the plasma membrane and shifts the transmembrane potential to more negative voltages. CNG channels are located exclusively in the outer segments of rods and cones. In darkness, a small fraction of the channels are in the open state, allowing an influx of cations. The intracellular concentration of Na + is low, whereas that of K + is high relative to the extracellular space, owing to the action of Na + /K + -ATPases in the inner segment. Thus, Na + and also some Ca 2+ ions flow in through the CNG channels in the outer segment. K + ions flow out through leak and voltage-gated ion channels in the inner segment to complete the electrical circuit, which is referred to as the “dark” or circulating current ( Fig. 19.2 ). Light absorbed by a visual pigment molecule in the outer segment closes CNG channels in an annulus of plasma membrane surrounding the site of photon absorption on the disk membrane.
When two photons are absorbed, it is unlikely that both absorptions occur in close proximity, so the response to each photon is generated independently and they summate. In other words, dim flashes lie within a linear operating range, wherein the response simply scales with the flash strength. However, with increasing numbers of photon absorptions, local effects begin to overlap, so fewer channels are closed per photon absorbed. With enough photons, all of the channels are closed, and the rod response is maximal ( Fig. 19.3 ). Hence, the normalized response grows with flash strength according to a saturating exponential function :
where r is the photocurrent response amplitude, r max is the amplitude of the maximal saturating response, i is flash strength, k f is a constant equal to ln(2)/i 0.5 , and i 0.5 is the flash strength giving rise to a half-maximal response. The i 0.5 for rods produces 30 to 70 photoisomerizations on average, whereas that for cones produces approximately 10 times as many photoisomerizations.
As is the case with photovoltage, photocurrent responses speed up with flash strength, although the effect is less pronounced in cones. , Responses are faster in cones than in rods (note the difference in time scales in Fig. 19.3A,C ) and have a marked undershoot (although the undershoot is controversial, compare , ). However, the photovoltage and photocurrent responses of rods and cones are not mirror images of each other. At least for red and green cones, the photovoltage response peaks slightly sooner than the photocurrent response, even for dim flashes. For rods, the dim flash photovoltage response peaks at about the same time as the photocurrent response, but the recovery is faster for the latter. In both rods and cones, there is a “nose” in the photovoltage response to brighter flashes ( Fig. 19.1 A,C ) that is absent from the photocurrent response ( Fig. 19.3 A,C ). , The basis for the nose is described further.
Once the flash is bright enough to close all the CNG channels, more light cannot produce a larger response, but instead, the duration of the photocurrent response increments with the natural logarithm of the flash strength ( Fig. 19.3D ). The basis for this behavior is that a single molecular process, namely the shutoff of transducin ( ; see Chapter 18 ), which has a stochastic, exponential time course, , dominates the recovery from phototransduction activation in rods. The slope of the saturation function or “Pepperberg Plot” yields the dominant time constant of about approximately 0.2 s (a similar value has been reported by many others for murine rods). The rate limiting step(s) in the recovery of the cone response has not been firmly established, but pigment quench is likely to contribute because transducin shutoff may be an order of magnitude faster than in rods. ,
Modulation of the flash response by bicarbonate
Bicarbonate is ubiquitous in the body and serves important roles in acid-base regulation and in providing a means to rid the body of metabolic CO 2 waste. In rods and cones, bicarbonate enlarges the dark current and accelerates flash response recovery , , by stimulating the membrane guanylate cyclases in a Ca 2+ -dependent fashion. Rods and cones take up bicarbonate through anion channels and transporters located at their synapses. Red and green cones, but not blue cones and rods, also synthesize bicarbonate internally because they express carbonic anhydrase. Bicarbonate diffuses throughout the cell, eventually exiting from the outer segments by way of a bicarbonate/chloride exchanger. , ,
Detection of single photons
Rods count single photons. To do so, they must minimize noise and generate highly amplified, reproducible quantal responses. Amplification is achieved by cascading several enzymatic reactions (see Chapter 18 ). Each chemical reaction takes time, hence the decrement in the dark current after photon absorption is delayed by a few milliseconds. Then the photocurrent response rises to a peak amplitude of 0.1 to 1 pA (this wide range is likely due to issues associated with experimental measurement and to species differences) approximately 200 ms later, reflecting the suppression of the dark current by a few percent. , , With the cascade working as fast as it can, the size of the response is determined by when shutoff and recovery processes kick in. In rods, hundreds of CNG channels are closed during the single photon response. Na + ions traverse the channel at a rate of approximately 10 4 per second (reviewed in ), so one photon blocks the entry of a million Na + ions. Responses to brighter flashes in rods and in cones appear with a shorter delay and rise more steeply because many more photoexcited rhodopsin molecules are activating the phototransduction machinery. In cones, the “single photon response” shuts off in 50 ms or less at a time when it has only reached an amplitude of a few tens of fA. ,
It is imperative that single photon responses do not vary in size or duration because if they did, the rod would be unable to distinguish one large, slow quantal response from two or more small, brief quantal responses occurring close together in time. When stimulated by dim flashes, the rod does not respond the same way every time, but most of the unpredictability arises from the Poisson distribution of photon absorptions, rather than from single photon response variability. Whenever the rod does respond, the amplitude is quantized ( Fig. 19.4A ). The coefficient of variation of the rod response, defined as the quotient of the standard deviation divided by the mean amplitude, has a low value of approximately 0.2. ,
In darkness, rods exhibit two physiologic kinds of electrical fluctuations in the current baseline: discrete noise and continuous noise ( Fig. 19.4B ). , Discrete noise is produced by thermal isomerizations of rhodopsin. Although rhodopsin has a half-life of approximately 400 years at body temperature, a rod contains so many copies (around a hundred million) that one spontaneously activates every couple of minutes. Continuous noise springs mainly from the spontaneous activity of phosphodiesterase (PDE) and is much more prevalent than discrete events but typically has a lower amplitude. The continuous noise amplitude distribution is approximately Gaussian, so occasionally there is a single photon response-like event. However, such events are inconsequential because they take place 10 times less frequently than the thermal isomerizations of rhodopsin. Although blue cone pigment may be even more thermally stable than rhodopsin, red and green cone visual pigments are far less stable. , Regardless, the overabundance of continuous noise, arising from gating transitions of the CNG channel, as well as from spontaneous activations of transducin and/or PDE, coupled with the low gain of phototransduction, preclude photon counting in cones.
Thermal isomerizations of rhodopsin confound the detection of very dim light because responses to real photons cannot be distinguished from “responses” to virtual ones. To guard against false alarms, the visual system relies on coincidence detection. A single photon response in one rod does not suffice for vision; a few rods in a small cluster within the retina must each generate a single photon response within a certain period of time before a flash is “seen.” The temporal requirement is specified by the integration time of the response. Thus, shortening the integration time reduces false alarms but detracts from sensitivity of the system. This consideration also applies when rods and cones respond to steps of light. The integration time for rods is approximately 300 ms, , , whereas for cones it is approximately 20 ms (segment preceding undershoot ).
Clearly, rods are well suited for single photon detection. Oddly though, rods sometimes generate single photon responses that are truly aberrant. , For reasons unknown, one photoexcited rhodopsin out of several hundred fails to shut off properly , and gives rise to a twofold larger than average response that can last for a very, very long time ( Fig. 19.5 ). Aberrant response durations are exponentially distributed with a mean value of about 4 s, but some last for tens of seconds. Aberrant responses have little impact on photon counting owing to their rarity and are not “seen” because the visual system requires several rods signaling the presence of a photon. However, they do improve sensitivity to steady light and by prolonging the recovery after exposure to bright light (flashes and steps), they leave rods vulnerable to saturation (see Box 19.1 ).
Excessive phototransduction cascade activity in photoreceptors can interfere with vision and lead to retinal disease (reviewed in ). For example, mutations that interfere with the normal shutoff of rhodopsin by targeting rhodopsin kinase (see Chapter 18 ) can cause a form of night blindness known as Oguchi disease. Mutant mouse rods lacking rhodopsin kinase generate aberrant single photon responses like those shown in Fig. 19.5 for every rhodopsin isomerization. Cones are only mildly affected in the disease, because they express a second type of rhodopsin kinase. , Patients with defects in the nuclear receptor NR2E3 develop enhanced S cone syndrome in which there is a higher prevalence of blue cones in the retina at the expense of red and green cones. Very interestingly, the blue cones in these patients fail to express either type of rhodopsin kinase. Hence, flash responses of their blue cones (but not red or green cones) take an abnormally long time to recover.
Mutations in arrestin, the protein normally responsible for quenching photoexcited rhodopsin’s activity, can also cause Oguchi disease (see Chapter 18 ). Flash responses from rods of mutant, arrestin knockout mice are very prolonged ( Fig. 19.8 ). Since longer lasting responses enhance absolute sensitivity, it might at first seem surprising that the mutations in either rhodopsin kinase or arrestin should lead to night blindness. The problem is that the mutant rods saturate at very low light levels and then take an inordinately long period of time and much dimmer conditions to recover from saturation. In theory, Oguchi patients with rhodopsin kinase or arrestin mutations might actually see better than normal persons under the dimmest conditions when given adequate time for dark adaptation. Photopic vision is largely spared because cones express a unique arrestin. ,
Mutations in either RGS9 or R9AP can sabotage the timely shutoff of transducin (see Chapter 18 ). Because these two proteins are used in rods and cones, there is night blindness and a problem with daytime vision. The ability of cones to light adapt enables them to escape saturation, so photopic vision is still possible. But long-lasting photoresponses translate into a disturbing persistence in sensation, and the person has difficulty following moving objects and adjusting to luminance changes, a condition termed bradyopsia.
Genetic defects in RPE65, the isomerase that converts all -trans to 11 -cis retinal (see Chapter 13 ), can prevent the de novo synthesis of rhodopsin. The persistent presence of catalytically active apo-opsin (rhodopsin lacking 11 -cis retinal is essentially equivalent to bleached rhodopsin) gives rise to Leber congenital amaurosis, an especially severe form of retinal degeneration. RPE65 knockout mouse rods exist in an inescapable state of light adaptation, with greatly attenuated dark current and smaller, faster flash responses ( Fig. 19.9 ).
Photocurrent response to steady light
The rod response to dim, steady light summates individual single photon responses. But with brighter light, adaptation within the outer segment causes the step responses to fall short of the amplitude expected from a simple saturation behavior ( , , ; see Chapter 20 ). Midrange responses droop as additional adaptational mechanisms with a slower time course reduce the cascade gain and cause the stimulus-response relation to rise even more gently ( Fig. 19.6 ). Light producing about 100 to 600 photoisomerizations per second decreases the dark current in darkness by 50% in rods of various primates. , , In contrast, the peak of the cone response to steps does almost obey simple saturation. A hundred milliseconds in the light later, however, cone responses begin to droop as they too adapt. For dim steps, the droop reduces the amplitude to less than half that at the peak. The initial droop is typically larger and faster in cones than in rods.
In steady light, incremental flashes give rise to responses in rods that are smaller ( Fig. 19.7 ) and recover more rapidly than in darkness, another manifestation of light adaptation ( , , , ; see Chapter 20 ). Flash response kinetics change very little with background light in primate cones, differing in this respect from cones of cold-blooded vertebrates. Light producing a few hundred photoisomerizations per second reduces flash sensitivity to half its value in darkness in rods, , , , where the value increases slowly over time in the light. , Adaptation is powerful in cones, for which 10,000 or more photoisomerizations per second reduce sensitivity twofold. , Mammalian rods were thought to adapt over several log units of background light intensity before saturating. , , However, recent studies found that mouse rods slowly adapt over tens of minutes to light levels previously considered to be purely photopic, and it seems likely that human rods share that capacity.
Light that is bright enough to bleach a significant fraction of visual pigment causes rods to behave for a period as if they were being exposed to a virtual, “equivalent light” that lingers after the light is removed (reviewed in ). Rods adapt to the equivalent light with a reduction in dark current and accelerated flash response kinetics, as well as a profound loss of flash sensitivity exceeding that expected from the decrease in photon capture (e.g., ). It turns out that bleached rhodopsin constitutively activates the phototransduction cascade. Although the activity of a single bleached rhodopsin is minuscule, the summated activity of hundreds of millions of bleached rhodopsin molecules is substantial. The equivalent light fades as visual pigment regenerates (see Chapter 13 and Box 19.1 ). Dark current after full bleach recovers halfway in 15 min and is fully restored in approximately 25 min. Cones are not impaired in this way by bleaching , ; their bleached pigment seems to be inactive (although the situation is not yet clear for bleached blue cone pigment, compare ). That means that when very bright light is turned on, cones may saturate, but they actually manage to recover circulating current during the exposure as loss of some of their visual pigment by bleaching lowers the ongoing rate of photon capture. Even after extensive bleaching, cones regain their full dark current and the reduction in sensitivity scales in proportion to the loss in photon capture. , , In the intact eye, cone pigment regeneration is faster than in rods, with full sensitivity returning after complete bleach within minutes. Therefore, whereas cones lack the absolute sensitivity of rods, they are better equipped to rapidly adapt over a wide range of brighter light intensities.
Action spectra of rods and cones
Rods and cones respond to a wide range of wavelengths ( Fig. 19.10 ), their action spectra being determined by the spectral absorptions of the visual pigment they express. For a given number of photons, the response amplitude varies with wavelength because the probability of absorption by the visual pigment varies with wavelength. The response itself to a photoisomerization is independent of wavelength. Rods respond maximally to a wavelength of approximately 493 nm (blue-green light). , There are three types of cones, commonly referred to as blue, green, and red with maxima near 430 nm (violet light), 530 nm (green light), and 560 nm (greenish-orange light), respectively ( , but see Box 19.2 ) ( Fig. 19.11 ). Since these cone names do not correspond to the colors of the maxima in every case, many prefer the designations short-wavelength sensitive or S, middle-wavelength sensitive or M, and long-wavelength sensitive or L.
Normal color vision requires the proper electrical signaling of light by all three cone types. Evolutionarily, the red and green cone pigment genes arose from a duplication event and they lie together on the X chromosome. Their high sequence similarity has caused “confusion” during crossing over in gametogenesis, creating within the population hybrid genes, fragmented genes, and variable numbers of copies of pigment genes on the chromosome ( Fig. 19.11 ). In persons with many genes in the array, expression of the first two predominates. Yet some of these individuals, and also women with different sets of pigment gene variants on their X chromosomes, actually possess more than three cone types in their retinas. The genetics help to explain the substantial individual variation in color vision in “normal” persons and anomalous color vision, as well as some forms of color “blindness” (reviewed in , see also Chapter 34 ). The last category includes extreme cases in whom failure to express one or both types of pigment genes on the X chromosome gives rise to dichromacy or blue cone monochromacy, respectively.
CNG channel and sodium/potassium/calcium exchanger
Ions move across the outer segment plasma membrane by two principal means: a CNG channel and a sodium/potassium/calcium exchanger. CNG channels are heterotetramers consisting of CNGA1 and CNGB1 subunits ( Fig. 19.12 ) in a ratio of 3:1 in rods and CNGA3 and CNGB3 subunits in a ratio of 3:1 in cones , (see Box 19.3 ). Although CNG channels are members of the same superfamily as voltage-gated K + channels, they are only mildly voltage-dependent in their gating (reviewed in ). Instead, CNG channels in rods and cones are directly gated by cGMP. , Cyclic AMP also works, but with a fiftyfold higher K 0.5 , reflecting the reduced affinity of the channel for cAMP and the reduced efficacy in opening once cAMP has bound. Whereas other ligand-gated ion channels in the body desensitize in the continued presence of their ligand, the CNG channel does not have any intrinsic mechanisms of desensitization and thus steadily monitors the [cGMP] in darkness. Channel sensitivity is subject to modulation under different conditions, for example, during light adaptation by calmodulin or a similar calcium-binding protein (see Chapter 20 ), by phosphorylation and by diacylglycerol.
