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W4256553683 abstract "Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Vertebrates acquired dim-light vision when an ancestral cone evolved into the rod photoreceptor at an unknown stage preceding the last common ancestor of extant jawed vertebrates (∼420 million years ago Ma). The jawless lampreys provide a unique opportunity to constrain the timing of this advance, as their line diverged ∼505 Ma and later displayed high-morphological stability. We recorded with patch electrodes the inner segment photovoltages and with suction electrodes the outer segment photocurrents of Lampetra fluviatilis retinal photoreceptors. Several key functional features of jawed vertebrate rods are present in their phylogenetically homologous photoreceptors in lamprey: crucially, the efficient amplification of the effect of single photons, measured by multiple parameters, and the flow of rod signals into cones. These results make convergent evolution in the jawless and jawed vertebrate lines unlikely and indicate an early origin of rods, implying strong selective pressure toward dim-light vision in Cambrian ecosystems. https://doi.org/10.7554/eLife.07166.001 eLife digest The eyes of humans and many other animals with backbones contain two different types of cells that can detect light, which are known as rod and cone cells. Rod cells are much more sensitive to light than cone cells. The rods allow us to see in dim light by amplifying weak light signals and transmitting information to other cells, including the cones themselves. It is thought that the rod cell evolved from the cone cell in the common ancestors of mammals, fish, and other animals with backbones and jaws at least 420 million years ago. Lampreys are jawless fish that diverged from the ancestors of jawed animals around 505 million years ago, in the middle of a period of great evolutionary innovation called the Cambrian. They have changed relatively little since that time so they provide a snapshot of what our ancestors' eyes might have been like back then. Like the rod and cone cells of jawed animals, the eyes of adult lampreys also have two types of photoreceptors. However, it was not clear whether the lamprey photoreceptor cells work in a similar way to rod and cone cells. Asteriti et al. collected lampreys in Sweden and France during their breeding season and used patch and suction electrodes to measure the activity of their photoreceptor cells. The experiments show that the short photoreceptor cells are more sensitive to light than the long photoreceptors and are able to amplify weak light signals. Also, the short photoreceptors send signals to the long photoreceptors in a similar way to how rod cells send information to cone cells. The similarities between lamprey photoreceptor cells and those of jawed animals support the idea that they have a common origin in evolutionary history. Therefore, Asteriti et al. conclude that the ability to see in low light evolved before these groups of animals diverged about 505 million years ago. The picture that emerges is one in which our remote ancestors inhabiting the Cambrian seas already possessed dim-light vision. This would have allowed them to colonize deep waters or to move at twilight, an adaptation suggestive of intense competition or predation from other life forms. https://doi.org/10.7554/eLife.07166.002 Introduction The fossil record shows that by the middle Cambrian, camera-type eyes were already present in stem vertebrates (Morris and Caron, 2014), supporting the emerging concept that spatially resolved vision provided a major competitive advantage in those biota (Paterson et al., 2011). Lampreys, the only surviving jawless vertebrates together with the related hagfish (Heimberg et al., 2010), are a pivotal resource for gaining further insight into early vertebrate vision. In fact, their line diverged during the Cambrian (∼505 Ma [Erwin et al., 2011]) and they later remained remarkably stable. This is true both of their external morphology, as revealed by fossil specimens (Janvier and Arsenault, 2002; Gess et al., 2006; Janvier et al., 2006; Chang et al., 2014), and of their anatomy, as demonstrated by primeval features such as the absence of bilateral limbs and of myelinated axons, and by their possession of the simplest nervous system among vertebrates. Adult lampreys have camera-type eyes with layered retinas containing all the major neuronal classes present in jawed vertebrates (Lamb, 2013) and sending retinotopically organized projections to the tectum (Jones et al., 2009), as well as a photosensory pineal organ (Pu and Dowling, 1981). Researchers have debated the rod or cone nature of lamprey retinal photoreceptors since the middle of the 19th century (relevant literature reviewed by Walls, 1935; Govardovskii and Lychakov, 1984; Collin et al., 2009) to ascertain whether, in vertebrates, cones pre-dated rods or vice versa. Current molecular genetic evidence indicates that modern rods evolved from an ancestral cone (Okano et al., 1992; Yokoyama, 2000; Lamb et al., 2007; Kawamura and Tachibanaki, 2008; Shichida and Matsuyama, 2009), implying that vision in near darkness is a relatively recent acquisition (Lamb, 2013) and causing the point of contention to become that of the timing of rod evolutionary emergence. This advance must have occurred (i) after the appearance of the precursor of rhodopsin and of other rod-specific phototransduction proteins isoforms and (ii) before the initial diversification of extant jawed vertebrates (∼420 Ma; Erwin et al., 2011) endowed with modern rods. Phylogenetic analysis of visual opsins constrains time bound i to have occurred anywhere between the divergence of ascidians (∼610 Ma; Erwin et al., 2011) and that of the lamprey line (∼505 Ma; Erwin et al., 2011): the sea squirt Ciona intestinalis has only one jawed vertebrate-related visual opsin (Kusakabe et al., 2001), while some lamprey species have all five major classes (Yokoyama, 2000) including an Rh1 rhodopsin ortholog (Pisani et al., 2006) (but see Collin et al., 2003). Recently, strong evidence has emerged indicating that these five opsin classes (and the rod-specific molecular toolbox) emerged in the context of two rounds of whole-genome duplication called ‘2R’ (Kuraku et al., 2009; Lagman et al., 2013). Furthermore, analysis of the whole sea lamprey genome suggests that the lamprey line diverged from the main vertebrate line shortly after 2R (Smith et al., 2013). Therefore, unveiling the functional properties of lamprey photoreceptors may shed light on the evolution of dim-light vision in the critical time period following 2R (Collin et al., 2009; Lamb, 2013). The two types of photoreceptors in the retina of Northern hemisphere lampreys have light-absorbing outer segments arranged in adjacent tiers (Figure 1A): those of short photoreceptors (SPs) lie in an inner tier, while those of long photoreceptors (LPs) lie in an outer tier, next to the pigment epithelium. This nomenclature is based on the entire length of the photoreceptors that of the outer segments showing instead the reverse pattern. Importantly, SPs express an Rh1 rhodopsin ortholog (Pisani et al., 2006) and some of their phototransduction protein isoforms examined thus far clade with those of rods (Muradov et al., 2008), but they also have molecular and morphological features of cones including outer segment discs that appear continuous with the plasma membrane (Dickson and Graves, 1979). Thus, while they retain archaic features of a cone progenitor, SPs are homologues of jawed vertebrate rods (Lamb, 2013). LPs, on the other hand, express an LWS red cone opsin and have a molecular fingerprint consistent with cones (Muradov et al., 2008). Here, we examined single lamprey photoreceptors at the levels of their inner and outer segments using two different recording techniques that provide complementary information, to establish the extent to which SPs operate like jawed vertebrate rods. We found multiple striking similarities that, taken together, argue against convergent evolution, implying that middle Cambrian vertebrates possessed functionally advanced rod precursors. Figure 1 with 1 supplement see all Download asset Open asset Signal processing in the inner segment of lamprey photoreceptors resembles that found in jawed vertebrates. (A) Image of a live retinal slice showing the layered organization of lamprey photoreceptors: short photoreceptors (SPs) in an inner tier and long photoreceptors (LPs) in an outer tier. Scale bar 10 µm. (B) Photoreceptors express the Ih current: membrane current of a SP in response to hyperpolarizing voltage clamp steps (from a holding potential of −53 mV to −60/−67/−74/−81/−88/−95/−102/−109 mV and repolarization to −65 mV) in control and during superfusion of the Ih blocker ZD7288 at 100 µM. Records are not averages. (C–F) Photovoltage responses reveal that SPs feed their signals into LPs. (C and D) Average responses to 520-nm flashes of a SP (0.5, 1.6, 5.4, 15, 45, 136, 398, 1128 photons·µm−2) and a LP (16, 51, 170, 469, 1413, 4314, 12,597, 38,847, 77,695 ph·µm−2). Insets show their outer segments (scale bars 5 µm). (E and F) Response amplitudes to 520-nm (green circles) and 590-nm flashes (orange circles with a dot) of a SP and a LP. Fits are exponential saturation functions (for the LP restricted to the first component: see text). In panel E, left and right ordinate values refer to left and right data sets, respectively, which were adjusted to match saturating amplitudes. In panel F such an adjustment could not be performed. Error bars are SEM. Action spectra templates for SPs and LPs are shown in Figure 1—figure supplement 1. https://doi.org/10.7554/eLife.07166.003 Results Using Lampetra fluviatilis, collected in Sweden and France during their spawning run, we investigated the function of photoreceptors in retinal slices maintained at a physiological temperature of 9–11°C. Dark membrane potentials and inner segment properties of SPs and LPs First, we made perforated patch-clamp recordings from photoreceptor inner segments and found that the dark membrane potential was of −43.2 ± 0.7 mV for SPs (n = 30) and −45.9 ± 1.1 mV for LPs (n = 10) (Table 1); these values are in line with those of jawed vertebrate rods and cones (Cangiano et al., 2012). Input resistances were 518 ± 41 MΩ (n = 8; SPs) and 442 ± 68 MΩ (n = 9; LPs). The membrane time constants, obtained by fitting single exponentials to the early rise of a current step response, were 31.9 ± 4.9 ms (n = 8; SPs) and 12.9 ± 1.3 ms (n = 9; LPs) (p < 0.001), equivalent to low-pass filtering with cut-off frequencies of ∼5 Hz for SPs and ∼12 Hz for LPs. Thus, the electrical properties of the inner segments of SPs seem adapted to process slower photocurrent changes than those of LPs. Both SPs (n = 7) and LPs (n = 2) expressed the hyperpolarization-activated current Ih, similarly to rods and cones (Della Santina et al., 2012); Ih was abolished by ZD7288 (100 µM, n = 1 SP; Figure 1B). Table 1 Electrophysiological parameters of SPs and LPs listed in the order they appear in the main text https://doi.org/10.7554/eLife.07166.010 ParameterPatch clampSuction electrodeSPsLPsSPsVdark (mV)−43.2 ± 0.7 (n = 30)−45.9 ± 1.1 (n = 10)–IRmembrane (MΩ)518 ± 41 (n = 8)442 ± 68 (n = 9)–τmembrane (ms)31.9 ± 4.9 (n = 8)[]a12.9 ± 1.3 (n = 9) [***]a–Max response (mv)3032–i1/2 (520 nm ph·µm−2); control149 ± 25 (n = 10) []b1.9 × 105 ± 1.1 × 105 (n = 7) []c–i1/2 (520 nm ph·µm−2); regenerated63 ± 11 (n = 12) [**]b2385 ± 513 (n = 7) [*]c–Sensitivity 520/590; control4.4 ± 0.9 (n = 5) []d1.1 ± 0.04 (n = 6) []e–Sensitivity 520/590; regenerated5.6 ± 1.0 (n = 5) [n.s.]d1.8 ± 0.1 (n = 7) [**]e–Integration time, dim flash (s); control0.32 ± 0.05 (n = 13) []f––Integration time, dim flash (s); regenerated0.81 ± 0.14 (n = 9) [***]f–1.45 ± 0.10 (n = 10)TTP at i1/2 (s); regenerated0.29 ± 0.04 (n = 11) []g0.11 ± 0.007 (n = 10) [***]g–τrec at i1/2 (s); regenerated1.05 ± 0.27 (n = 11) []h0.12 ± 0.02 (n = 10) [***]h–i1/2 (λmax ph·µm−2); regenerated63 ± 11 (n = 12) []i777 ± 167 (n = 7) [***]i–Dim-flash sensitivity (mV·ph−1·µm2); regenerated0.61 ± 0.17 (n = 8)––Dim-flash sensitivity (%·ph−1·µm2); regenerated3.0 ± 0.6 (n = 8)––a (pA); regenerated––0.41 ± 0.04 (n = 10)a% (%·R* −1); regenerated2.6 ± 0.5 (n = 8)–2.6 ± 0.3 (n = 10)SNR; regenerated––1.5 ± 0.1 (n = 10)Idark (pA); regenerated13 ± 3 (n = 4)–16 ± 1 (n = 10)Collecting area (µm2·R*·ph−1); regenerated––0.83 ± 0.17 (n = 10)Amplification constant (s−2); regenerated––0.59 ± 0.09 (n = 10) Values are given as ‘mean ± SEM (sample size) [statistical significance]identifier letter’; n.s.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001; Vdark: dark membrane potential; IRmembrane: input resistance; τmembrane: membrane time constant; i1/2: half-maximal response flash strength; TTP: time-to-peak; τrec: decay time constant; a: absolute single photon response; a%: fractional single photon response; SNR: signal-to-noise ratio; Idark: dark current; SPs: short photoreceptors; LPs: long photoreceptors. SPs feed their signals to LPs Light stimulation evoked a hyperpolarization in both photoreceptors (Figure 1C,D), with peak changes in membrane potential of up to 30 mV (SPs) and 32 mV (LPs) in response to saturating flashes. The amplitudes of the flash responses from SPs were described by exponential saturation functions (Figure 1C,E). From the curves, we obtained a ratio of 4.4 ± 0.9 (n = 5) for the sensitivities of these photoreceptors at 520 nm and 590 nm. This value is in reasonably good agreement with the ratio of 5.6 predicted by an 11A1 visual pigment template (Govardovskii et al., 2000) having a λmax of 517 nm (Figure 1—figure supplement 1A), the absorbance maximum of SP outer segments found with microspectrophotometry (Govardovskii and Lychakov, 1984), and is thus consistent with the expression of an Rh1 visual pigment. For LPs, the flash responses displayed two components (Figure 1D,F): the first component had kinetics, sensitivity, and spectral preference similar to SPs; the second component had faster kinetics, lower sensitivity, and a ratio of sensitivities at 520 nm and 590 nm of 1.1 ± 0.04 (n = 6). This ratio agrees with the value of 1.1 predicted by an 11A1 template (Govardovskii et al., 2000), whose λmax is set at 555 nm (Figure 1—figure supplement 1A), the absorbance maximum of LP outer segments (Govardovskii and Lychakov, 1984), consistent with their expression of an LWS pigment. It is likely that the first component of the flash response from LPs represents input from SPs, probably mediated by gap junctions; this arrangement would represent in lamprey retina an arrangement homologous to rod-cone coupling in jawed vertebrates (Asteriti et al., 2014). In support of this interpretation, we observed with Lucifer Yellow injection thin telodendria emanating from the synaptic pole of the photoreceptors and extending laterally into the inner plexiform layer (Figure 2): the only known function of these processes in jawed vertebrates is that of forming inter-photoreceptor junctional contacts (O'Brien et al., 2012). We attempted to uncouple these cells pharmacologically with MFA (100 µM) or 2-APB (10–20 µM), known blockers of retinal gap junctions, but unfortunately these agents produced marked non-specific effects (not shown). Figure 2 Download asset Open asset Lamprey photoreceptors extend telodendrial processes. An example of a lucifer yellow stain of a live SP showing two thin processes (arrowheads) extending laterally from the synaptic pole into the outer plexiform layer. https://doi.org/10.7554/eLife.07166.005 SPs display bleaching desensitization During their spawning run, lampreys do not feed and rely exclusively on stored reserves for several months, leading to a vitamin A deficiency (Wald, 1942) that could hinder visual pigment regeneration. We thus wondered whether some of the visual pigment in our preparations might have been in a bleached state (i.e., devoid of its light-sensing chromophore). Clarifying this point was a crucial prerequisite to our subsequent assessment of single photon processing by SPs, for reasons explained in the rest of this paragraph. In jawed vertebrates, bleached rod and cone opsins constitutively activate the phototransductive cascade at a very low rate (Cornwall and Fain, 1994; Cornwall et al., 1995). Due to this property in rods, in which pigment regeneration is much slower than in cones, bleaches of even a small fraction of the total pigment pool caused by bright light lead to a significant and long-lasting desensitization, which is much larger than what is expected from the simple decrease in light-sensitive visual pigment molecules (Fain et al., 2001; Lamb and Pugh, 2004). Bleaching desensitization thus leads to a reduction in phototransduction gain, and therefore, in the single photon response amplitude. Assuming that lamprey opsins behave similarly to those of jawed vertebrates, the possible presence of bleached visual pigment in our experiments (see above) raises the possibility that SPs were desensitized relative to their full potential. To examine whether this was the case, we regenerated any bleached visual pigment molecules by superfusing the retinal slices in the recording chamber with the artificial analog 9-cis-Retinal (100 µM for 20–25 min). The sensitivity at 520 nm of two SPs, recorded both in control and during delivery of 9-cis-Retinal, increased by 2.0 and 2.5-fold (Figure 3A). Moreover, sensitivity was higher (p < 0.01) in regenerated than in control SPs: half-maximal response at 520 nm evoked with flashes (i1/2) of 63 ± 11 photons·µm−2 (n = 12; regenerated) vs 149 ± 25 photons·µm−2 (n = 10; control). Thus, some of the visual pigment molecules in SP outer segments were indeed bleached. The ratio of sensitivities at 520 and 590 nm did not differ significantly (p = 0.42) between regenerated and control SPs: 5.6 ± 1.0 (n = 5; regenerated) vs 4.4 ± 0.9 (n = 5; control). To test whether such bleaching was associated to desensitization, we examined dim-flash integration time (Jones et al., 1996). In one SP, integration time increased by 2.6-fold after superfusion with 9-cis-Retinal and the same parameter was significantly higher (p < 0.001) in regenerated than in control SPs (Figure 3B): 0.81 ± 0.14 s (n = 9; regenerated) vs 0.32 ± 0.05 s (n = 13; control). These results strongly suggest that SPs were in a state of bleaching desensitization. Figure 3 Download asset Open asset Visual pigment regeneration reveals the full sensitivity of photoreceptors in the upstream migrating river lamprey. (A) Photovoltage response amplitudes to 520-nm flashes before (gray circles) and after visual pigment regeneration with 9-cis-Retinal (red circles with a dot) of a SP. Left and right ordinate values refer to left and right data sets, respectively, which were adjusted to match saturating amplitudes. (B) Normalized-averaged-normalized dim-flash photovoltage responses in control (n = 13) and regenerated SPs (n = 9), highlighting the difference in integration time. These records were obtained as follows: (i) the average dim-flash response of each SP was normalized to its peak amplitude (always below 2 mV), (ii) normalized responses were averaged across cells, (iii) the final average was normalized to its peak. Shaded areas show ±1 SEM. (C) Photovoltage response amplitudes to 520-nm flashes before (gray circles) and after visual pigment regeneration with 9-cis-Retinal (red circles with a dot) of a LP. Responses to 590-nm flashes are also shown (small empty circles; error bars are smaller than circle diameter). Error bars are SEM. https://doi.org/10.7554/eLife.07166.006 Although secondary to the main goal of this analysis, we also tested the effect of 9-cis-Retinal on LPs. In one LP, the sensitivity at 520 nm increased by 22-fold after superfusion with 9-cis-Retinal (Figure 3C; second, lower sensitivity component). Moreover, sensitivity was much higher (p < 0.05) in regenerated than in control LPs: half-maximal response at 520 nm evoked with flashes (i1/2) of 2385 ± 513 photons·µm−2 (n = 7; regenerated) vs 1.9 × 105 ± 1.1 × 105 photons·µm−2 (n = 7; control; second, lower sensitivity component; see ‘Materials and methods’ for details on the uncertainty of this specific value). As expected for the incorporation of 9-cis-Retinal in a significant fraction of the LP visual pigment pool (Makino et al., 1999), this increase in sensitivity was associated with a marked hypsochromic shift. Specifically, the ratio of sensitivities at 520 and 590 nm went from 1.1 to 1.7 in the single LP treated with 9-cis-Retinal (Figure 3C, compare large and small circles) and was significantly higher (p < 0.01) in regenerated than in control LPs: 1.8 ± 0.1 (n = 7; regenerated) vs 1.1 ± 0.04 (n = 6; control). To examine the properties of lamprey photoreceptors under fully dark-adapted conditions, all subsequent experiments were made on retinas pretreated with 9-cis-Retinal. SPs are intrinsically slower than LPs After pigment regeneration, the photovoltage responses of SPs, recorded with patch clamp, remained markedly slower than those of LPs (Figure 4A,B). To characterize the photoreceptors' kinetics, for each recorded cell we plotted time-to-peak (TTP) and decay time constant (τrec) as a function of flash strength normalized to its half-maximal value (i1/2) (Figure 4C,D). From linear fits to the data, we estimated the values of these parameters at i1/2: for SPs, the TTP was 0.29 ± 0.04 s (n = 11) and the τrec was 1.05 ± 0.27 s (n = 11). In contrast, for LPs, the TTP was only 0.11 ± 0.007 s (n = 10; p < 0.001) and the τrec only 0.12 ± 0.02 (n = 10; p < 0.001). Therefore, when compared at flash strengths eliciting responses of similar fractional amplitude, SPs were indeed slower than LPs. Importantly, while the estimates of TTP and τrec in LPs may have been influenced to some degree by the signals that are fed to them from SPs (see above), the latter would have acted to reduce (rather than increase) the differences in kinetics between the two photoreceptors. Figure 4 Download asset Open asset SPs are markedly slower than LPs. (A and B) Average responses to 520-nm flashes of a SP (0.5, 1.6, 5.4, 15, 45, 136, 398, 1128 photons·µm−2) and a LP (16, 51, 170, 469, 1413, 4314, 12,597, 38,847, 77,695 ph·µm−2), both recorded with patch clamp after visual pigment regeneration with 9-cis-Retinal. (C and D) Plots of time-to-peak (TTP) and decay time constant (τrec) vs flash strength, normalized to its half-maximal value i1/2, in regenerated SPs (n = 11; empty circles) and LPs (n = 10; full circles). The data from each cell are connected by lines. https://doi.org/10.7554/eLife.07166.007 SPs are intrinsically more sensitive than LPs To compare the intrinsic light sensitivity of regenerated SPs and LPs, we first considered whether we should correct their half-maximal flash strengths (i1/2) measured at 520 nm for: (i) the position of the peaks of their action spectra (λmax) with respect to the stimulus wavelength and (ii) the smaller quantum efficiency of pigment bound to 9-cis-Retinal (about one third; Hubbard and Kropf, 1958; Hurley et al., 1977). Both factors have the effect of reducing the sensitivity displayed by the photoreceptor with respect to its maximum achievable level. For SPs, we made the conservative assumption that they incorporated only a negligible amount of 9-cis-Retinal, as suggested by their limited increase in sensitivity following regeneration combined with their expression of bleaching desensitization (and supported by the non-significant change in their 520/590 nm sensitivity ratios). This implied that our 520-nm flashes essentially coincided with λmax (517 nm, see above) and that no correction was necessary for their i1/2 of 63 ± 11 photons·µm−2 (n = 12). Given the conservative nature of the above assumption, this value provides a lower bound for the maximal sensitivity of fully dark-adapted SPs. For LPs, we made the equally conservative assumption that their entire visual pigment pool was replaced with 9-cis-Retinal. We then predicted their modified action spectrum (Figure 1—figure supplement 1B) by slightly adjusting two parameters of the 9A1 template for red cones of Makino et al. (1999) (λmax_A0 from 508 to 507 nm and λmax_G1 from 567 to 566 nm; see their Table 2) so as to match our experimentally determined ratio of sensitivities at 520 and 590 nm of 1.8 ± 0.1 (n = 7; see above). Taking into account the off-peak position of our flashes (520 nm) relative to the λmax of this action spectrum (541 nm) and the smaller quantum efficiency of regenerated pigment, the corrected i1/2 of LPs was 777 ± 167 photons·µm−2 (n = 7). Given the conservative initial assumption, this value provides an upper bound for the maximal sensitivity of fully dark-adapted LPs. The i1/2 of LPs was much higher than that of SPs (p < 0.001). Note that signals feeding from SPs into LPs would have acted to reduce (rather than increase) the differences in sensitivity between the two photoreceptors, leaving our conclusions unchanged. The single photon response of SPs is within the range of jawed vertebrate rods A crucial functional measure of the position of lamprey SPs with respect to the evolutionary transition from an ancestral cone to the modern rod is their performance in amplifying single photons (Lamb, 2013). Regenerated SPs were highly sensitive, with absolute and fractional dim-flash sensitivities in patch clamp of 0.61 ± 0.17 mV·photons−1·µm2 (n = 8) and 3.0 ± 0.6%·photons−1·µm2 (n = 8). We obtained a first estimate of their fractional single photon response of 2.6 ± 0.5%·R* −1 (n = 8) by dividing the fractional dim-flash sensitivity with a theoretical effective collecting area of 1.18 µm2·R*·photons−1 (see ‘Materials and methods’). A search for quantal responses using the patch-clamp technique proved inconclusive, as also observed in similar recordings of photoreceptors from those jawed vertebrates having extensively coupled rods (Fain, 1975). We thus performed suction electrode photocurrent recordings from the conical outer segments of SPs, in retinae pretreated with 9-cis-Retinal (100 µM for 20–25 min): this recording technique only measures the current flowing through the membrane enclosed in the pipette and is thus ideally suited to examine phototransduction in a given photoreceptor without an appreciable contribution of its electrically coupled neighbors (Baylor et al., 1979a). Under these conditions, responses to repeated delivery of dim flashes were highly variable in amplitude (Figure 5A). We estimated the absolute amplitude of the single photon response (a) to be 0.41 ± 0.04 pA (n = 10), by dividing the increase in the time-dependent variance by the mean response for each SP (Figure 5B) (Rieke and Baylor, 1998) (for details on the use of variance analysis in single photon response estimation see the ‘Materials and methods’). The normalized time-dependent squared mean responses and variance increases overlapped (Figure 5B) (Rieke and Baylor, 1998), consistent with the single photon responses being governed by Poisson statistics. The fractional amplitude of the single photon response (a%), determined for each SP on the basis of its maximal response to a single saturating flash delivered prior to the dim flash trains, was 2.6 ± 0.3%·R* −1 (n = 10), in line with the independent estimate obtained with patch (see above). Lastly, the signal-to-noise ratio (SNR), determined for each SP as the ratio of a over the standard deviation of the biological component of dark noise measured between consecutive dim flashes (0.5–20 Hz), was 1.5 ± 0.1 (n = 10). Importantly, the values of a, a%, and SNR in SPs are within the range reported for jawed vertebrate rods (Figure 6). In these experiments, we: (i) recorded only from intact outer segments (Figure 1C, inset), (ii) observed similar dark currents (maximum current change with a saturating flash) with patch clamp (13 ± 3 pA, n = 4) and suction electrodes (16 ± 1 pA, n = 10), and (iii) measured similar collecting areas (0.83 ± 0.17 µm2·R*·photons−1, n = 10; ratio of the squared mean response over the product of the variance increase and the flash strength [Rieke and Baylor, 1998]) to theoretical prediction (1.18 µm2·R*·photons−1). From this, it is highly likely that we had complete suction of the outer segment, and as such, we could make reliable estimates of a% and SNR. Figure 5 Download asset Open asset Suction electrode recordings of SP photocurrents in the single photon regime. (A) Samples of dim-flash response trains recorded from the outer segments of 4 SPs (SP1: flash strength = 1.64 photons·µm−2, a = 0.25 pA; SP2: 3.26 ph·µm−2, 0.43 pA; SP3: 1.64 ph·µm−2, 0.56 pA; SP4: 3.26 ph µm−2, 0.56·pA). (B) Single photon response analysis from one SP (SP3 in panel A), showing the mean response μ (thin trace: gross mean response, dotted trace: mean dark current, thick trace: net mean response), time-dependent variance σ2 (thin trace: gross variance, dotted trace: dark current variance, thick trace: net variance), normalized squared mean response norm μ2 (thin trace) and variance norm σ2 (thick trace). Dashed lines indicate the current baseline or zero level. Dark current records were taken from the last 2 s preceding each flash and where baselined in the first 1.1 s, there" @default.
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W4256553683 date "2015-03-17" @default.
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W4256553683 title "Decision letter: A Cambrian origin for vertebrate rods" @default.
W4256553683 doi "https://doi.org/10.7554/elife.07166.011" @default.
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