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Endogenous nitric oxide enhances the light-response of cones during light-adaptation in the rat retina
Vision Research 51 (2011) 131–137
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Endogenous nitric oxide enhances the light-response of cones during light-adaptation in the rat retina Masaki Sato a, Teruya Ohtsuka a,⇑, William K. Stell b,c,d a
Biology Research Division, Graduate School of Science, Toho University, 2-2-1 Miyama, Funabashi, 274-8510 Chiba, Japan Department of Cell Biology and Anatomy, University of Calgary Faculty of Medicine, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1 c Division of Ophthalmology, Department of Surgery, University of Calgary Faculty of Medicine, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1 d Hotchkiss Brain Institute, University of Calgary Faculty of Medicine, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1 b
a r t i c l e
i n f o
Article history: Received 18 February 2010 Received in revised form 23 September 2010
Keywords: CPTIO Electroretinogram Cone photoreceptor Rat Light-adaptation
a b s t r a c t The electroretinogram (ERG) is a non-invasive indicator of retinal function. Light flashes evoke a corneanegative a-wave followed by a cornea-positive b-wave. Light-adaptation is known to increase the amplitude of cone-dependent b-waves. To identify the underlying mechanism, we recorded rat cone photoresponses in situ, using intravitreally-injected glutamate to block synaptic transmission and intense pairedflash stimuli to isolate cone a-waves. Steady adapting illumination caused a progressive increase in cone a-wave amplitude, which was suppressed in a dose-dependent manner by intravitreal CPTIO, a nitric oxide scavenger. We conclude that light-adaptation causes release of nitric oxide, which enhances the cone photoresponse. Ó 2010 Published by Elsevier Ltd.
1. Introduction The electroretinogram (ERG) is a mass retinal light-response, due to the activity of many retinal cells (Gouras, 2005). Its major components are a cornea-negative a-wave (from photoreceptor cells) and a cornea-positive b-wave (primarily from second-order neurons). The b-wave amplitude has been shown to increase gradually for several minutes after onset of a sustained adapting light (Alexander, Raghuram, & Rajagopalan, 2006; Gouras & MacKay, 1989; Peachey, Alexander, Fishman, & Derlacki, 1989), in the human ERG (Burian, 1954) and the ERGs of non-human species, including monkey (Murayama & Sieving, 1992), rat (Bui & Fortune, 2006), and mouse (Peachey, Goto, al-Ubaidi, & Naash, 1993). The underlying mechanism, however, remains unknown. Recently, we found that intravitreal injection of an exogenous nitric oxide (NO) donor, SNAP, exerted opposite effects on rod and cone components of the rat ERG, suppressing the light-evoked response of rods but enhancing that of cones (Sato & Ohtsuka, 2010). This suggested that NO regulates the saturation-level of rod signals in retinal ON and OFF pathways – before the synaptic endings of cone bipolar cells, where rod and cone signals converge (Protti, FloresHerr, Li, Massey, & Wässle, 2005), and even before the synapses of photoreceptors onto bipolar cells, where NO has been reported
⇑ Corresponding author. Fax: +81 474 72 5257 E-mail address: [email protected] (T. Ohtsuka). 0042-6989/$ - see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.visres.2010.10.011
to modulate synaptic transmission (Kourennyi et al., 2004; Kurenny, Moroz, Turner, Sharkey, & Barnes, 1994; Savchenko, Barnes, & Kramer, 1997). In the previous study, however, we were not able to show the predicted effect of an intravitreally-injected NO scavenger, CPTIO, on isolated rod or cone ERGs in dark-adapted rats (Sato & Ohtsuka, 2010). In the present study, we have tested the hypothesis that endogenous NO release is minimal in the dark-adapted state but elevated in the early stages of light-adaptation (Hoshi, Sato, Oguri, & Ohtsuka, 2003), and that this light-elicited increase in NO mediates light-evoked changes in the responses of cones themselves. To do this, we determined whether CPTIO inhibits the effects of light-adaptation on cones, thus revealing the activation of nitrergic mechanisms during intense light-adaptation as suggested elsewhere (Eldred, 2001; Sekaran, Cunningham, Neal, Hartell, & Djamgoz, 2005). We developed a protocol for isolating the cone component of the rat ERG, using intravitreal glutamate injection to block signal transmission between photoreceptors and second-order neurons (Sato & Ohtsuka, 2010) and an intense paired-flash technique to isolate cone photoresponses (Weymouth & Vingrys, 2008). We found that intravitreal CPTIO suppresses the light adaptation-induced increase in amplitude of isolated cone photoresponses in a dose-dependent manner. This indicates for the first time that increases in the rate of synthesis and release of NO in the retina, due to light-adaptation, cause progressive increases in the light-responses of cone photoreceptors themselves.
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2. Material and methods 2.1. Animals Male Sprague–Dawley rats, age 8–10 weeks, were kept under a fluorescent room lamp (lights on 08:00–20:00). For ERG measurements, rats were kept in darkness for 2 h prior to the experiment, to obtain consistent results for both rod and cone ERGs, then anesthetized by intraperitoneal injection of 25% urethane (#050-05821, Wako, Osaka, Japan), 6 mL/kg, and the head was fixed in a stereotaxic frame (SG-3, Narishige, Tokyo, Japan). Body temperature was maintained at 38 °C. These procedures were done under a dim red safelight. After the experiments, the rats were kept for a week under normal vivarium conditions, to verify that the ERG recovered fully and that no adverse effects on their cornea and lens could be detected under the binocular microscope, and then they were euthanized with diethyl ether. Experimental procedures were approved by the Toho University Animal Research Ethics Committee. 2.2. Drug administration For isolating the photoreceptor component, we injected intravitreally 2 lL of 0.1–1.0 M monosodium glutamate (#138-14945, Wako) dissolved in 0.1 M phosphate-buffered saline (PBS), pH 7.2, under anesthesia. The solution was delivered via the 26-gauge needle of a Hamilton microsyringe, inserted 2 mm posterior to the superior limbus. The resulting ‘intravitreal concentration of glutamate’ (hereafter: [Glu]) was calculated as 4–40 mM, assuming 47 lL as the vitreous volume in the rat (Sha & Kwong, 2006). NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline1-oxyl-3-oxide (CPTIO; #C348, Dojindo, Kumamoto, Japan), was diluted in PBS to 1, 10 and 100 lM in PBS. The mixed solution, 1 lL of one of these CPTIO solutions + 1 lL of 0.4 M glutamate, was injected intravitreally. The ‘final concentration of CPTIO in the vitreous cavity’ (hereafter: [CPTIO]) was calculated as 0.04, 0.4 and 4 lM, and [Glu] was 8 mM. CPTIO removes active NO, converting it to NO2 and NO3 (Akaike et al., 1993). Because of its high affinity for NO, CPTIO antagonizes guanylyl cyclase-activation, and thus NO-activated cGMP-synthesis (Crawford et al., 2006). CPTIO is accepted as a specific scavenger for free-radical NO (Akaike et al., 1993; Yoshida et al., 1994) and is effective at concentrations of 10–300 lM in rat tissues in vitro (Rand & Li, 1995). 2.3. ERG recording After corneal application of 1% atropine sulfate (#019-04851, Wako) for sustained dilation of the pupil, and 0.4% oxybuprocaine hydrochloride (#B0750, Santen, Osaka, Japan) for local anesthesia, bundled stainless steel–fiber loop electrodes, 6 mm outer diameter, were placed on the left cornea as active electrode and on the right cornea as reference, with a ground-electrode in the oral cavity (Sato & Ohtsuka, 2010). ERGs were recorded with a bioamplifier (MEZ-8201, Nihon Kohden, Tokyo, Japan), with the band-pass set to 0.3–1 kHz. ERG and stimulus signals were digitized via a 12bit A/D board (ADM-682PCI, Microscience, Chiba, Japan) and stored in a personal computer. The 1 kHz sampling frequency and the storage mode were controlled by programmable software (LabDAQ-Pro, Matsuyama Advance, Ehime, Japan). 2.4. Light-stimulation Light stimuli from two green LEDs (kmax = 530 nm; Luxeon Star/O LXHL-NM98, Philips Lumileds, San Jose, CA, USA) were delivered to the left eye. One, the intense adapting light, was for inducing NO release from retinal cells (Hoshi et al., 2003), while the other, the
double flash, was for suppressing the rod-derived component (Sato & Ohtsuka, 2010). The double-flash LED was delivered through a light-guide (LG-SF, Olympus, Tokyo, Japan), illuminating a field 9 mm in diameter at the cornea, while the adapting light LED was placed near the light-guide outlet. Stimulus irradiance, determined with a photometer (J16, Tektronix, Tokyo, Japan), was 5.6 lW/cm2 for the adapting light and 700 lW/cm2 for the double flash at the corneal surface. A pulse-generator (SEN-6100, Nihon Kohden) produced pairs of 10-ms flashes, separated by 1 s, once per minute. Because rods were still desensitized 1 s after the first flash, the second flash elicited a pure-cone response. Ten successive pure-cone responses were averaged under each experimental condition for quantitative study. 2.5. Statistical analysis Effects of the drugs were expressed quantitatively as the ratio, ‘amplitude of isolated cone ERG recorded after drug-injection’ to ‘amplitude of corresponding cone ERG recorded before drug-injection’. Results for each experimental condition are expressed as the ratios from four-five rats (mean ± standard deviation). Statistical analysis was by Dunnett’s post hoc test, and P < 0.05 was taken as significant. 3. Results 3.1. Dependence of light-adapted ERG on concentration and time after administration of glutamate Before using glutamate to isolate the mass photoreceptor response in situ, we studied the time-course of action of glutamate at different concentrations ([Glu] = 4–40 mM) in five groups of rats, using the paired-flash ERG paradigm (two 10-ms flashes, separated by 1 s). Our previous experiments showed that it took more than 30 min to block the rod-derived ERG b-wave, but less than 5 min to block the cone-derived ERG b-wave (Sato & Ohtsuka, 2010). In the present study, therefore, an intense paired flash (700 lW/ cm2) was superposed on a less intense but still strongly photopic adapting light (5.6 lW/cm2), which also maintained the release of NO. Using this combination of light stimuli, we observed recovery of the ERG after 60 min when [Glu] was less than 8 mM. The test flash was delivered after 1 min of light-adaptation, just before the adapting background light was turned off. In order to maintain a constant state of light-adaptation during ERG testing, we provided a 1-min rest-period in the dark before onset of the next 1-min adapting light. Both a- and b-wave amplitudes were constant when sampled this way, once during every 2-min light–dark cycle (Fig. 1). Intravitreal injection of glutamate blocked glutamatergic transmission between photoreceptors and bipolar cells, thus causing a rapid decrease in amplitude of the cone-derived ERG b-wave (hereafter called ‘‘cone b-wave’’) to zero within 610 min. This led to the emergence of a cone-derived ERG a-wave (hereafter called ‘‘cone a-wave’’), having a maximum amplitude of 20–25 lV, which had been obscured by the larger b-wave. Vehicle alone (PBS) had no effect, and the lowest [Glu] caused a small (<20%) decline in the cone b-wave and no change in the cone a-wave. At higher concentrations, recovery time was a function of [Glu]; isolation of the cone a-wave was maintained for 31 min at [Glu] = 8 mM and for 61 min at [Glu] = 20 mM, with loss of cone a- and b-wave isolation and recovery of a complete, normal ERG (vehicle control) after 661 min and 67 days, respectively. After raising the intravitreal concentration to [Glu] = 40 mM, the cone a-wave reached a maximum amplitude within 5 min, but then declined rapidly, and the ERG never recovered. The decline in cone b-wave amplitude
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3.2. Increase of cone-ERG amplitude by light-adaptation As reported previously (Bui & Fortune, 2006; Sato & Ohtsuka, 2010), rat ERGs elicited in situ by intense paired-flash stimuli were bimodal, consisting of a fast, cornea-negative a-wave followed by a slower, larger cornea-positive b-wave (Fig. 2B, 1–2). In the absence of drug treatments, the first flash elicited an ERG containing both rod and cone components, while the second flash elicited an ERG containing only cone components (Fig. 2B, 1–2, 6–7). Intense adapting light suppressed the rod component and the a-wave, leaving only the cone-dependent b-wave (Fig. 2B, 3–5), as shown previously (Bui & Fortune, 2006). After onset of an adapting light, the amplitudes of the cone ERG b-waves to both flashes gradually increased, reaching a maximum after about 8 min; this timecourse is consistent with light-induced changes in human and other mammalian cone ERG b-waves (noted earlier). 0.5 min after cessation of the adapting light, the first flash elicited a mixed rodcone ERG (Fig. 2B, 6); after 30 min, the amplitudes of ERGs evoked by both flashes had recovered to about 80% of initial levels (Fig. 2B, 7), and after another 30 min, they had both recovered fully (result not shown, but indistinguishable from Fig. 2B, 1). Intravitreal 8 mM [Glu] dramatically altered the form of the ERG during steady light-adaptation (Fig. 2C), abolishing the conederived ERG b-wave (the cone component: response to second flash) within 10 min (Fig. 2C, 2), while the rod-derived ERG b-wave (the rod component: response to first flash) remained unaffected for 30 min (Fig. 2C, 6). However, since the rod component was abolished by the intense first flash, we could analyze the cone component in isolation, as it is precisely the glutamate-isolated ERG evoked by the second flash. The light-adapted cone ERG shown here is a pure cornea-negative a-wave, and therefore mainly a response of cones themselves. The amplitude of this isolated cone awave increased gradually for the first several minutes of lightadaptation (Fig. 2C, 3–4) and then reached a steady plateau (Fig. 2C, 5). After termination of the adapting light, the ERG amplitudes gradually recovered (Fig. 2C, 6–7), returning to normal within 2 h in all animals. Inspection of the eyes with an operating microscope 1 week after treatment did not reveal any adverse effects on their corneas or lenses. 3.3. Effect of CPTIO on isolated cone ERG
Fig. 1. Dependence of light-adapted ERG on glutamate concentration and time after injection. (A) Upper trace: the cone-derived ERG b-wave after injection of vehicle only, recorded just after glutamate injection and onset of light-adaptation (at time 0); arrow indicates amplitude of the b-wave, measured from the baseline. Lower trace: the cone-derived ERG a-wave isolated by [Glu] = 8 mM, recorded 10 min after onset of light-adaptation. The a-wave inside the rectangle is shown at 2.5 enlargement in the inset. Visible AC noise superposed on the cone a-wave, indicated by a filled arrowhead, was removed by averaging ten successive traces for the quantitative analysis shown in C. (B) Glutamate-dependence of amplitude of conederived ERG b-wave (relative response amplitude at stated time after injection, as percent of amplitude before injection). Calculated intravitreal concentration of glutamate [Glu], shown by different symbols (mean ± SD; n = 4), is shown at the far right. (C) [Glu]-dependence of amplitude of the cone-derived ERG a-wave (absolute amplitude, measured from baseline). Symbols are the same as in (B).
paralleled the rise in cone a-wave amplitude, except that after [Glu] = 40 mM there was still no ERG b-wave, even after recovery for 1 week. These data indicated that recording glutamate-isolated cone ERGs 10–20 min after raising the intravitreal [Glu] to 8 mM provides a stable baseline for assessing the effects of other agents on glutamate-isolated mass cone responses.
The nitric oxide (NO) scavenger, CPTIO, was used to test whether the effects of intense light-adaptation on cone photoresponses are mediated by the light-evoked release of NO in the retina. CPTIO exerted no apparent effect on either rod or cone ERGs for the first 2 h, after which the ERG amplitude decreased gradually for a day or so (Sato & Ohtsuka, 2010). Treatment with [Glu] = 8 mM + [CPTIO] = 0.04 lM caused no apparent changes in ERGs (Fig. 3A, 1–7) compared to those recorded after the same amount of glutamate alone (Fig. 2C). In contrast, [Glu] = 8 mM + [CPTIO] = 0.4 lM suppressed the light adaptation-induced increase in amplitude of cone photoresponses by about half (Fig. 3B, 5), and [Glu] = 8 mM + [CPTIO] = 4.0 lM abolished that increase completely (Fig. 3C, 5). Thus, intravitreal CPTIO suppressed the lightinduced increase in amplitude of cone photoresponses, in a dose-dependent manner. The time-courses of changes in cone-ERG amplitude after onset of the adapting light, and the effects of different intravitreal concentrations of CPTIO, are summarized in Fig. 4. Since the absolute amplitude of the recorded ERG varied from one animal to another, mainly because of variations in electrode placement, maximum b-wave amplitudes varied from 650 to 900 lV in the five rats studied. Therefore, amplitudes were normalized to an internal standard, the amplitude of the b-wave before drug-injection (see Section 2). The amplitude of the isolated cone photoresponse
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two-thirds of the control amplitude and did so only after 13 min; and adding [CPTIO] = 4 lM completely abolished any detectable increase in amplitude during the observation period. The dosedependent differences in effects of CPTIO were already significant (P < 0.05) only 2 min after onset of the adapting light, and they increased further over time (Fig. 4).
4. Discussion
Fig. 2. (A) Scheme of the experimental procedure: numbers at the top indicate times when ERGs shown in (B) and (C) were obtained. The upper trace shows time after injection of glutamate (at time zero); and the lower trace shows the time-course of the adapting light, which was on from 10 to 30 min after injection. (B) After control injection: ERGs to paired-flash stimulus without chemical agents. In #1 and #2: Before onset of the adapting light, the first flash elicited mixed rod-cone ERG and the second a pure-cone ERG. Just after the onset of light-adaptation (#3), and 5 min (#4) and 10 min (#5) later, both flashes elicited pure-cone ERGs. In #6 and #7: ERGs 0.5 and 30 min after light-off show recovery. (C) After glutamate injection: #1: Control; the upward arrow indicates the amplitude of ERG b-wave before glutamate injection. #2: After injecting Glu to produce [Glu] = 8 mM, but 0.5 min before onset of the adapting light, ERG b-wave to the second flash was completely suppressed. #3–5: Glutamate-isolated cone ERGs, averaged 10, with 2.5-expanded voltage axis. The amplitudes of these cone ERGs were quantitatively studied in Fig. 4. #6 and #7 (0.5 and 30 min, respectively, after light-off). ERGs within rectangle were analyzed quantitatively (see details in text). Time- and voltage-calibration bars were the same in (B) and (C), except in (C) #3–5, in which the voltage axis is expanded 2.5 (calibration = 0.2 mV).
increased steadily with time after onset of the adapting light, reaching a maximum after 8 min (Fig. 4, filled circles). In the absence of drug treatment, the light-adapted second-flash ERG bwave (enclosed by the rectangles in Fig. 2B, 3–5) also recovered with a similar time-course, as reported previously in human and other animal species (Alexander et al., 2006; Bui & Fortune, 2006; Burian, 1954; Gouras & MacKay, 1989; Murayama & Sieving, 1992; Peachey, Alexander et al., 1989, Peachey, Goto et al., 1993). We found that the time-course of these adaptation-dependent changes in cone photoresponse was the same after raising [Glu] to 8 mM by injecting Glu alone, or adding [CPTIO] = 0.04 lM To [Glu] = 8 mM. In contrast, the addition of [CPTIO] = 0.4 lM slowed and diminished the increase in amplitude, which reached only
These results indicate that glutamate, administered intraocularly at a sub-toxic dose, produces stable isolation of photoreceptor responses for 20 min in the living, anesthetized rat, and they confirm that pure cone-derived responses can be isolated by means of strong light-adaptation and intense double-flash ERG techniques, as we have shown previously (Sato & Ohtsuka, 2010). Our results obtained using this method indicate that CPTIO, administered intraocularly in an appropriate dose range (Yoshida et al., 1994), inhibits a physiological process that selectively and progressively enhances the cone photoresponse during the first minutes of adaptation to intense light. Because CPTIO is a scavenger of free-radical NO (Akaike et al., 1993; Rand & Li, 1995; Yoshida et al., 1994), this implies that the synthesis and release of NO from retinal cells are increased by intense ambient illumination (Ding & Weinberg, 2007; Eldred, 2001; Neufeld, Shareef, & Pena, 2000) and that CPTIO inhibits the light-responses of cone photoreceptors by removing NO-dependent stimulation of their photoresponses during the early stages of light-adaptation. Burian (1954) was the first to report that the amplitude of the human ERG b-wave increases over time under intense adapting light. In the five decades since then, a similar phenomenon has been observed by many others, in human (Alexander et al., 2006; Peachey, Alexander et al., 1989) as well as other vertebrate ERGs (Bui & Fortune, 2006; Murayama & Sieving, 1992; Peachey, Goto et al., 1993). However, the underlying mechanism of this phenomenon, which might seem at first to contradict the desensitization of cone responses that is expected in light-adaptation (Gouras & MacKay, 1989), is not yet known. The cone membrane potential in the fish retina depolarizes back toward the dark membrane potential under intense adapting light (Burkhardt & Gottesman, 1987). This slow depolarization over time acts to increase the amplitude of the cone photoresponse to additional flashes, in turn causing the amplitude of the cone ERG b-wave also to increase gradually as light-adaptation progresses (Gouras & MacKay, 1989). The results of the present study suggest that NO is released from retinal cells during the initial stages of light-adaptation (Ding & Weinberg, 2007; Eldred, 2001; Neufeld et al., 2000; Sekaran et al., 2005) and that this endogenous NO is a major adaptational regulator of cone photoreceptor activity. Although we found no effect of CPTIO on glutamate-isolated cone ERGs in a previous study (Sato & Ohtsuka, 2010), in that study we did not employ strong light-adaptation, and the condition of the retina was almost fully dark-adapted. Under those conditions, the generation and release of endogenous NO, and therefore also the effect of CPTIO, should have been minimal, as we reported (Sato & Ohtsuka, 2010). From this we may conclude that the release of NO is not involved in the sensitization of rod photoresponses during dark-adaptation. How might NO elicit the effects reported here? Some insight may be derived from recent studies in several model systems, showing that NO can open Ca2+-channels, thereby increasing intercellular Ca2+ concentration and modulating the properties of numerous membrane receptors and channels (Almanza, Navarrete, Vega, & Soto, 2007; Ko & Kelly, 1999; Kourennyi et al., 2004). Such actions require a complete signaling cascade, consisting of: NO source + mediator + effector (NOS – soluble guanylyl cyclase (sGC) – cyclic guanosine monophosphate (cGMP) – cyclic
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Fig. 3. Effect on cone ERGs, of adding different amounts of intravitreal CPTIO to [Glu] = 8 mM. Numbers at the far left correspond to the times when ERGs were sampled, as shown in Fig. 2A (top row). In #1: Control, upward arrow indicates the ERG b-wave amplitude before glutamate + CPTIO injection. Amplitudes of isolated cone ERGs (#3–#5) were averaged 10 before analyzing quantitatively, because they were so small. (A) At lowest dose of CPTIO, isolated cone ERGs (#3–#5) were the same as those after the same amount of glutamate only, as in Fig. 1C. (B) At intermediate dose of CPTIO, cone ERGs (#3–#5) increased less during light-adaptation than in (A). (C) At highest dose of CPTIO, cone ERGs were minimal and amplitudes did not increase during light-adaptation. Time and voltage-calibration bars were the same for (A–C), except in #3–#5, in which the voltage axis is expanded 2.5 (calibration = 0.2 mV).
Fig. 4. Time-course of changes in glutamate-isolated and adaptation-modified cone ERGs, after injection of CPTIO. Filled circles (data from Fig. 2C) represent the control, the ratio of ‘amplitude of cone ERG after application of [Glu] = 8 mM’ to ‘amplitude of ERG b-wave before injection’. The open symbols (data from Fig. 3A–C) represent the same ratio after simultaneous injection of different amounts of CPTIO, shown as the calculated intravitreal concentrations [CPTIO]. The ordinate represents that ratio as percent. Each point and error bar represent the mean ± SD, for n = 5 rats.
nucleotide-gated (CNG) channel), or direct actions of NO, e.g., via Snitrosylation or Tyr-nitration of proteins. Actions of NO on synaptic transmission, which have been well documented in other studies (e.g., Kourennyi et al., 2004), could not have been responsible for the effects that we observed in Glu-treated retinas, in which synaptic transmission from photoreceptors was inactivated. Therefore, we have to seek explanations by mechanisms located in, or closely associated with, the non-synaptic regions of rods and cones. Most authors report that the molecular components of the NOactivated cascade are absent from outer segments, in rat (Kajimura et al., 2003; Kim et al., 1999; López-Costa, Goldstein, & Pecci Saavedra, 1997; Neufeld et al., 2000; Shin et al., 1999; Yamamoto, Bredt, Snyder, & Stone, 1993) as in other species (Blom, Blute, & Eldred, 2009; Haberecht et al., 1998; Koch, Lambrecht, Haberecht, Redburn, & Schmidt, 1994; Neufeld et al., 2000). In any case,
membrane-bound GC, the predominant form and the source of cGMP for transduction in outer segments, is not activated by the NO-donor, sodium nitroprusside (Margulis, Sharma, & Sitaramayya, 1992). Photoreceptor inner segments, however, do contain all the machinery for generating as well as responding to NO – in particular, the NO-synthesizing enzyme, NOS (Haberecht et al., 1998; Kim et al., 1999; Koch et al., 1994; Kurenni et al., 1995; Kurenny et al., 1994; López-Costa et al., 1997; Neufeld et al., 2000; Shin et al., 1999; Yamamoto et al., 1993), and a cGMP target, the cGMP-gated ion channel (Matthews & Watanabe, 1988; Watanabe & Matthews, 1988). The intermediary cGMP-producing enzyme, sGC, a prime NO target, has been detected in inner segments by some investigators (Koch et al., 1994; Nakazawa et al., 2005; Zhang, Beuve, & Townes-Anderson, 2005), but not by others (Kajimura et al., 2003; Blom et al., 2009; Haberecht et al., 1998), perhaps because of problems with antibody cross-reactivity. The rather stout, strongly sGC-immunoreactive rodlike structures in the rat retina, designated outer segments by Nakazawa et al. (2005), may be inner segments, since they appear to arise from the external limiting membrane. The present results are consistent with our previous observation that increasing retinal NO concentration, by intravitreal injection of an exogenous NO-donor (SNAP), suppressed the rod ERG a-wave but enhanced the cone ERG a-wave (Sato & Ohtsuka, 2010). Thus NO is a ‘two-faced’ modulator of retinal function, in that NO released by retinal neurons in response to intense adapting light suppresses the light-evoked activity of (and/or synaptic transmission from) rod photoreceptors, while enhancing the corresponding functions of cone photoreceptors. Such push–pull antagonism not only would provide an ambient-light-dependent mechanism for switching rapidly between rod and cone systems at the input stage to the retina; but under mesopic conditions it could also prevent saturation of inner-retinal signal transmission in those ON and OFF pathways where rod and cone signals converge (Protti et al., 2005). How the cGMP-gated channel and other ion channels regulate cytoplasmic free calcium concentration
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[Ca2+] in inner segments, and how NO might affect rod- and conedependent ERG components in opposite ways, remain unknown, but Zhang et al. (2005) discuss several possibilities. In salamander retina the voltage-dependent calcium-activated currents, IK(Ca) and ICl, were identified electrophysiologically in rod and cone inner segments (Bader, Bertrand, & Schwartz, 1982); a NO-donor, S-nitrosocysteine (SNC), was found to increase a Ca2+-channel current (Kurenny et al., 1994); cGMP enhanced calcium influx into cones, but not into rods (Rieke & Schwartz, 1994); SNC enhanced the depolarization-induced increases of [Ca2+] in rods, but inhibited them in cones (Kourennyi et al., 2004); and NO, working through cGMP and cGMP-gated channels, led to a calcium-induced increase of neurotransmitter release from cones (Savchenko et al., 1997). Plasma-membrane Ca2+-ATPase was reported to extrude Ca2+ more rapidly from cones than from rods (Krizaj, Lai, & Copenhagen, 2003). Since Ca2+ modulates many cytoplasmic signaling cascades and binds to guanylyl cyclase-activating proteins 1 and 2, which are distributed unequally between cones and rods in mammals (Cuenca, Lopez, Howes, & Kolb, 1998), differences in both nucleotide- and Ca2+-mediated signaling may be involved in the differential responses of cones and rods to NO and functional state. Previous studies, utilizing the ERG (Alexander et al., 2006; Bui & Fortune, 2006; Burian, 1954; Gouras & MacKay, 1989; Murayama & Sieving, 1992; Peachey, Alexander et al., 1989; Peachey, Goto et al., 1993; Sato & Ohtsuka, 2010) and single-cell recording (Kourennyi et al., 2004), have shown that NO regulates synaptic transmission from cones to second-order neurons. The present results reveal another, unexpected major role of NO in the retina, which is to regulate (at a stage prior to synaptic transmission) the photoresponses of cones themselves. Finally, differential responses of rods and cones to a wide variety of agents and stimulus conditions appear to be the rule, not the exception, and we have now added nitric oxide as an important differential regulator of light- and dark-adaptation in photoreceptor cells.
5. Conclusion In the present study, we isolated the cone component of the rat ERG (cone photoresponse) by a combination of intravitreal glutamate injection and intense paired-flash stimulation. Under intense adapting light, the amplitude of the isolated cone photoresponse increased gradually, reaching a maximum after about 8 min. This increase was suppressed in a dose-dependent manner by intravitreal injection of the NO scavenger, CPTIO. Our results suggest that NO-synthesis and -release from retinal neurons are enhanced by exposure to relatively intense (photopic) illumination, and that the increase in endogenous NO readjusts cone gain and/or sensitivity during the initial stages of light-adaptation. Since CPTIO is reported to be a specific NO scavenger at the doses used in the present study, it is logical to conclude that NO is the major messenger used by the retina to restore cone signaling rapidly after shifts to higher, and initially saturating, ambient light intensities.
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Vision Research journal homepage: www.elsevier.com/locate/visres
Endogenous nitric oxide enhances the light-response of cones during light-adaptation in the rat retina Masaki Sato a, Teruya Ohtsuka a,⇑, William K. Stell b,c,d a
Biology Research Division, Graduate School of Science, Toho University, 2-2-1 Miyama, Funabashi, 274-8510 Chiba, Japan Department of Cell Biology and Anatomy, University of Calgary Faculty of Medicine, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1 c Division of Ophthalmology, Department of Surgery, University of Calgary Faculty of Medicine, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1 d Hotchkiss Brain Institute, University of Calgary Faculty of Medicine, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1 b
a r t i c l e
i n f o
Article history: Received 18 February 2010 Received in revised form 23 September 2010
Keywords: CPTIO Electroretinogram Cone photoreceptor Rat Light-adaptation
a b s t r a c t The electroretinogram (ERG) is a non-invasive indicator of retinal function. Light flashes evoke a corneanegative a-wave followed by a cornea-positive b-wave. Light-adaptation is known to increase the amplitude of cone-dependent b-waves. To identify the underlying mechanism, we recorded rat cone photoresponses in situ, using intravitreally-injected glutamate to block synaptic transmission and intense pairedflash stimuli to isolate cone a-waves. Steady adapting illumination caused a progressive increase in cone a-wave amplitude, which was suppressed in a dose-dependent manner by intravitreal CPTIO, a nitric oxide scavenger. We conclude that light-adaptation causes release of nitric oxide, which enhances the cone photoresponse. Ó 2010 Published by Elsevier Ltd.
1. Introduction The electroretinogram (ERG) is a mass retinal light-response, due to the activity of many retinal cells (Gouras, 2005). Its major components are a cornea-negative a-wave (from photoreceptor cells) and a cornea-positive b-wave (primarily from second-order neurons). The b-wave amplitude has been shown to increase gradually for several minutes after onset of a sustained adapting light (Alexander, Raghuram, & Rajagopalan, 2006; Gouras & MacKay, 1989; Peachey, Alexander, Fishman, & Derlacki, 1989), in the human ERG (Burian, 1954) and the ERGs of non-human species, including monkey (Murayama & Sieving, 1992), rat (Bui & Fortune, 2006), and mouse (Peachey, Goto, al-Ubaidi, & Naash, 1993). The underlying mechanism, however, remains unknown. Recently, we found that intravitreal injection of an exogenous nitric oxide (NO) donor, SNAP, exerted opposite effects on rod and cone components of the rat ERG, suppressing the light-evoked response of rods but enhancing that of cones (Sato & Ohtsuka, 2010). This suggested that NO regulates the saturation-level of rod signals in retinal ON and OFF pathways – before the synaptic endings of cone bipolar cells, where rod and cone signals converge (Protti, FloresHerr, Li, Massey, & Wässle, 2005), and even before the synapses of photoreceptors onto bipolar cells, where NO has been reported
⇑ Corresponding author. Fax: +81 474 72 5257 E-mail address: [email protected] (T. Ohtsuka). 0042-6989/$ - see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.visres.2010.10.011
to modulate synaptic transmission (Kourennyi et al., 2004; Kurenny, Moroz, Turner, Sharkey, & Barnes, 1994; Savchenko, Barnes, & Kramer, 1997). In the previous study, however, we were not able to show the predicted effect of an intravitreally-injected NO scavenger, CPTIO, on isolated rod or cone ERGs in dark-adapted rats (Sato & Ohtsuka, 2010). In the present study, we have tested the hypothesis that endogenous NO release is minimal in the dark-adapted state but elevated in the early stages of light-adaptation (Hoshi, Sato, Oguri, & Ohtsuka, 2003), and that this light-elicited increase in NO mediates light-evoked changes in the responses of cones themselves. To do this, we determined whether CPTIO inhibits the effects of light-adaptation on cones, thus revealing the activation of nitrergic mechanisms during intense light-adaptation as suggested elsewhere (Eldred, 2001; Sekaran, Cunningham, Neal, Hartell, & Djamgoz, 2005). We developed a protocol for isolating the cone component of the rat ERG, using intravitreal glutamate injection to block signal transmission between photoreceptors and second-order neurons (Sato & Ohtsuka, 2010) and an intense paired-flash technique to isolate cone photoresponses (Weymouth & Vingrys, 2008). We found that intravitreal CPTIO suppresses the light adaptation-induced increase in amplitude of isolated cone photoresponses in a dose-dependent manner. This indicates for the first time that increases in the rate of synthesis and release of NO in the retina, due to light-adaptation, cause progressive increases in the light-responses of cone photoreceptors themselves.
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2. Material and methods 2.1. Animals Male Sprague–Dawley rats, age 8–10 weeks, were kept under a fluorescent room lamp (lights on 08:00–20:00). For ERG measurements, rats were kept in darkness for 2 h prior to the experiment, to obtain consistent results for both rod and cone ERGs, then anesthetized by intraperitoneal injection of 25% urethane (#050-05821, Wako, Osaka, Japan), 6 mL/kg, and the head was fixed in a stereotaxic frame (SG-3, Narishige, Tokyo, Japan). Body temperature was maintained at 38 °C. These procedures were done under a dim red safelight. After the experiments, the rats were kept for a week under normal vivarium conditions, to verify that the ERG recovered fully and that no adverse effects on their cornea and lens could be detected under the binocular microscope, and then they were euthanized with diethyl ether. Experimental procedures were approved by the Toho University Animal Research Ethics Committee. 2.2. Drug administration For isolating the photoreceptor component, we injected intravitreally 2 lL of 0.1–1.0 M monosodium glutamate (#138-14945, Wako) dissolved in 0.1 M phosphate-buffered saline (PBS), pH 7.2, under anesthesia. The solution was delivered via the 26-gauge needle of a Hamilton microsyringe, inserted 2 mm posterior to the superior limbus. The resulting ‘intravitreal concentration of glutamate’ (hereafter: [Glu]) was calculated as 4–40 mM, assuming 47 lL as the vitreous volume in the rat (Sha & Kwong, 2006). NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline1-oxyl-3-oxide (CPTIO; #C348, Dojindo, Kumamoto, Japan), was diluted in PBS to 1, 10 and 100 lM in PBS. The mixed solution, 1 lL of one of these CPTIO solutions + 1 lL of 0.4 M glutamate, was injected intravitreally. The ‘final concentration of CPTIO in the vitreous cavity’ (hereafter: [CPTIO]) was calculated as 0.04, 0.4 and 4 lM, and [Glu] was 8 mM. CPTIO removes active NO, converting it to NO2 and NO3 (Akaike et al., 1993). Because of its high affinity for NO, CPTIO antagonizes guanylyl cyclase-activation, and thus NO-activated cGMP-synthesis (Crawford et al., 2006). CPTIO is accepted as a specific scavenger for free-radical NO (Akaike et al., 1993; Yoshida et al., 1994) and is effective at concentrations of 10–300 lM in rat tissues in vitro (Rand & Li, 1995). 2.3. ERG recording After corneal application of 1% atropine sulfate (#019-04851, Wako) for sustained dilation of the pupil, and 0.4% oxybuprocaine hydrochloride (#B0750, Santen, Osaka, Japan) for local anesthesia, bundled stainless steel–fiber loop electrodes, 6 mm outer diameter, were placed on the left cornea as active electrode and on the right cornea as reference, with a ground-electrode in the oral cavity (Sato & Ohtsuka, 2010). ERGs were recorded with a bioamplifier (MEZ-8201, Nihon Kohden, Tokyo, Japan), with the band-pass set to 0.3–1 kHz. ERG and stimulus signals were digitized via a 12bit A/D board (ADM-682PCI, Microscience, Chiba, Japan) and stored in a personal computer. The 1 kHz sampling frequency and the storage mode were controlled by programmable software (LabDAQ-Pro, Matsuyama Advance, Ehime, Japan). 2.4. Light-stimulation Light stimuli from two green LEDs (kmax = 530 nm; Luxeon Star/O LXHL-NM98, Philips Lumileds, San Jose, CA, USA) were delivered to the left eye. One, the intense adapting light, was for inducing NO release from retinal cells (Hoshi et al., 2003), while the other, the
double flash, was for suppressing the rod-derived component (Sato & Ohtsuka, 2010). The double-flash LED was delivered through a light-guide (LG-SF, Olympus, Tokyo, Japan), illuminating a field 9 mm in diameter at the cornea, while the adapting light LED was placed near the light-guide outlet. Stimulus irradiance, determined with a photometer (J16, Tektronix, Tokyo, Japan), was 5.6 lW/cm2 for the adapting light and 700 lW/cm2 for the double flash at the corneal surface. A pulse-generator (SEN-6100, Nihon Kohden) produced pairs of 10-ms flashes, separated by 1 s, once per minute. Because rods were still desensitized 1 s after the first flash, the second flash elicited a pure-cone response. Ten successive pure-cone responses were averaged under each experimental condition for quantitative study. 2.5. Statistical analysis Effects of the drugs were expressed quantitatively as the ratio, ‘amplitude of isolated cone ERG recorded after drug-injection’ to ‘amplitude of corresponding cone ERG recorded before drug-injection’. Results for each experimental condition are expressed as the ratios from four-five rats (mean ± standard deviation). Statistical analysis was by Dunnett’s post hoc test, and P < 0.05 was taken as significant. 3. Results 3.1. Dependence of light-adapted ERG on concentration and time after administration of glutamate Before using glutamate to isolate the mass photoreceptor response in situ, we studied the time-course of action of glutamate at different concentrations ([Glu] = 4–40 mM) in five groups of rats, using the paired-flash ERG paradigm (two 10-ms flashes, separated by 1 s). Our previous experiments showed that it took more than 30 min to block the rod-derived ERG b-wave, but less than 5 min to block the cone-derived ERG b-wave (Sato & Ohtsuka, 2010). In the present study, therefore, an intense paired flash (700 lW/ cm2) was superposed on a less intense but still strongly photopic adapting light (5.6 lW/cm2), which also maintained the release of NO. Using this combination of light stimuli, we observed recovery of the ERG after 60 min when [Glu] was less than 8 mM. The test flash was delivered after 1 min of light-adaptation, just before the adapting background light was turned off. In order to maintain a constant state of light-adaptation during ERG testing, we provided a 1-min rest-period in the dark before onset of the next 1-min adapting light. Both a- and b-wave amplitudes were constant when sampled this way, once during every 2-min light–dark cycle (Fig. 1). Intravitreal injection of glutamate blocked glutamatergic transmission between photoreceptors and bipolar cells, thus causing a rapid decrease in amplitude of the cone-derived ERG b-wave (hereafter called ‘‘cone b-wave’’) to zero within 610 min. This led to the emergence of a cone-derived ERG a-wave (hereafter called ‘‘cone a-wave’’), having a maximum amplitude of 20–25 lV, which had been obscured by the larger b-wave. Vehicle alone (PBS) had no effect, and the lowest [Glu] caused a small (<20%) decline in the cone b-wave and no change in the cone a-wave. At higher concentrations, recovery time was a function of [Glu]; isolation of the cone a-wave was maintained for 31 min at [Glu] = 8 mM and for 61 min at [Glu] = 20 mM, with loss of cone a- and b-wave isolation and recovery of a complete, normal ERG (vehicle control) after 661 min and 67 days, respectively. After raising the intravitreal concentration to [Glu] = 40 mM, the cone a-wave reached a maximum amplitude within 5 min, but then declined rapidly, and the ERG never recovered. The decline in cone b-wave amplitude
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3.2. Increase of cone-ERG amplitude by light-adaptation As reported previously (Bui & Fortune, 2006; Sato & Ohtsuka, 2010), rat ERGs elicited in situ by intense paired-flash stimuli were bimodal, consisting of a fast, cornea-negative a-wave followed by a slower, larger cornea-positive b-wave (Fig. 2B, 1–2). In the absence of drug treatments, the first flash elicited an ERG containing both rod and cone components, while the second flash elicited an ERG containing only cone components (Fig. 2B, 1–2, 6–7). Intense adapting light suppressed the rod component and the a-wave, leaving only the cone-dependent b-wave (Fig. 2B, 3–5), as shown previously (Bui & Fortune, 2006). After onset of an adapting light, the amplitudes of the cone ERG b-waves to both flashes gradually increased, reaching a maximum after about 8 min; this timecourse is consistent with light-induced changes in human and other mammalian cone ERG b-waves (noted earlier). 0.5 min after cessation of the adapting light, the first flash elicited a mixed rodcone ERG (Fig. 2B, 6); after 30 min, the amplitudes of ERGs evoked by both flashes had recovered to about 80% of initial levels (Fig. 2B, 7), and after another 30 min, they had both recovered fully (result not shown, but indistinguishable from Fig. 2B, 1). Intravitreal 8 mM [Glu] dramatically altered the form of the ERG during steady light-adaptation (Fig. 2C), abolishing the conederived ERG b-wave (the cone component: response to second flash) within 10 min (Fig. 2C, 2), while the rod-derived ERG b-wave (the rod component: response to first flash) remained unaffected for 30 min (Fig. 2C, 6). However, since the rod component was abolished by the intense first flash, we could analyze the cone component in isolation, as it is precisely the glutamate-isolated ERG evoked by the second flash. The light-adapted cone ERG shown here is a pure cornea-negative a-wave, and therefore mainly a response of cones themselves. The amplitude of this isolated cone awave increased gradually for the first several minutes of lightadaptation (Fig. 2C, 3–4) and then reached a steady plateau (Fig. 2C, 5). After termination of the adapting light, the ERG amplitudes gradually recovered (Fig. 2C, 6–7), returning to normal within 2 h in all animals. Inspection of the eyes with an operating microscope 1 week after treatment did not reveal any adverse effects on their corneas or lenses. 3.3. Effect of CPTIO on isolated cone ERG
Fig. 1. Dependence of light-adapted ERG on glutamate concentration and time after injection. (A) Upper trace: the cone-derived ERG b-wave after injection of vehicle only, recorded just after glutamate injection and onset of light-adaptation (at time 0); arrow indicates amplitude of the b-wave, measured from the baseline. Lower trace: the cone-derived ERG a-wave isolated by [Glu] = 8 mM, recorded 10 min after onset of light-adaptation. The a-wave inside the rectangle is shown at 2.5 enlargement in the inset. Visible AC noise superposed on the cone a-wave, indicated by a filled arrowhead, was removed by averaging ten successive traces for the quantitative analysis shown in C. (B) Glutamate-dependence of amplitude of conederived ERG b-wave (relative response amplitude at stated time after injection, as percent of amplitude before injection). Calculated intravitreal concentration of glutamate [Glu], shown by different symbols (mean ± SD; n = 4), is shown at the far right. (C) [Glu]-dependence of amplitude of the cone-derived ERG a-wave (absolute amplitude, measured from baseline). Symbols are the same as in (B).
paralleled the rise in cone a-wave amplitude, except that after [Glu] = 40 mM there was still no ERG b-wave, even after recovery for 1 week. These data indicated that recording glutamate-isolated cone ERGs 10–20 min after raising the intravitreal [Glu] to 8 mM provides a stable baseline for assessing the effects of other agents on glutamate-isolated mass cone responses.
The nitric oxide (NO) scavenger, CPTIO, was used to test whether the effects of intense light-adaptation on cone photoresponses are mediated by the light-evoked release of NO in the retina. CPTIO exerted no apparent effect on either rod or cone ERGs for the first 2 h, after which the ERG amplitude decreased gradually for a day or so (Sato & Ohtsuka, 2010). Treatment with [Glu] = 8 mM + [CPTIO] = 0.04 lM caused no apparent changes in ERGs (Fig. 3A, 1–7) compared to those recorded after the same amount of glutamate alone (Fig. 2C). In contrast, [Glu] = 8 mM + [CPTIO] = 0.4 lM suppressed the light adaptation-induced increase in amplitude of cone photoresponses by about half (Fig. 3B, 5), and [Glu] = 8 mM + [CPTIO] = 4.0 lM abolished that increase completely (Fig. 3C, 5). Thus, intravitreal CPTIO suppressed the lightinduced increase in amplitude of cone photoresponses, in a dose-dependent manner. The time-courses of changes in cone-ERG amplitude after onset of the adapting light, and the effects of different intravitreal concentrations of CPTIO, are summarized in Fig. 4. Since the absolute amplitude of the recorded ERG varied from one animal to another, mainly because of variations in electrode placement, maximum b-wave amplitudes varied from 650 to 900 lV in the five rats studied. Therefore, amplitudes were normalized to an internal standard, the amplitude of the b-wave before drug-injection (see Section 2). The amplitude of the isolated cone photoresponse
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two-thirds of the control amplitude and did so only after 13 min; and adding [CPTIO] = 4 lM completely abolished any detectable increase in amplitude during the observation period. The dosedependent differences in effects of CPTIO were already significant (P < 0.05) only 2 min after onset of the adapting light, and they increased further over time (Fig. 4).
4. Discussion
Fig. 2. (A) Scheme of the experimental procedure: numbers at the top indicate times when ERGs shown in (B) and (C) were obtained. The upper trace shows time after injection of glutamate (at time zero); and the lower trace shows the time-course of the adapting light, which was on from 10 to 30 min after injection. (B) After control injection: ERGs to paired-flash stimulus without chemical agents. In #1 and #2: Before onset of the adapting light, the first flash elicited mixed rod-cone ERG and the second a pure-cone ERG. Just after the onset of light-adaptation (#3), and 5 min (#4) and 10 min (#5) later, both flashes elicited pure-cone ERGs. In #6 and #7: ERGs 0.5 and 30 min after light-off show recovery. (C) After glutamate injection: #1: Control; the upward arrow indicates the amplitude of ERG b-wave before glutamate injection. #2: After injecting Glu to produce [Glu] = 8 mM, but 0.5 min before onset of the adapting light, ERG b-wave to the second flash was completely suppressed. #3–5: Glutamate-isolated cone ERGs, averaged 10, with 2.5-expanded voltage axis. The amplitudes of these cone ERGs were quantitatively studied in Fig. 4. #6 and #7 (0.5 and 30 min, respectively, after light-off). ERGs within rectangle were analyzed quantitatively (see details in text). Time- and voltage-calibration bars were the same in (B) and (C), except in (C) #3–5, in which the voltage axis is expanded 2.5 (calibration = 0.2 mV).
increased steadily with time after onset of the adapting light, reaching a maximum after 8 min (Fig. 4, filled circles). In the absence of drug treatment, the light-adapted second-flash ERG bwave (enclosed by the rectangles in Fig. 2B, 3–5) also recovered with a similar time-course, as reported previously in human and other animal species (Alexander et al., 2006; Bui & Fortune, 2006; Burian, 1954; Gouras & MacKay, 1989; Murayama & Sieving, 1992; Peachey, Alexander et al., 1989, Peachey, Goto et al., 1993). We found that the time-course of these adaptation-dependent changes in cone photoresponse was the same after raising [Glu] to 8 mM by injecting Glu alone, or adding [CPTIO] = 0.04 lM To [Glu] = 8 mM. In contrast, the addition of [CPTIO] = 0.4 lM slowed and diminished the increase in amplitude, which reached only
These results indicate that glutamate, administered intraocularly at a sub-toxic dose, produces stable isolation of photoreceptor responses for 20 min in the living, anesthetized rat, and they confirm that pure cone-derived responses can be isolated by means of strong light-adaptation and intense double-flash ERG techniques, as we have shown previously (Sato & Ohtsuka, 2010). Our results obtained using this method indicate that CPTIO, administered intraocularly in an appropriate dose range (Yoshida et al., 1994), inhibits a physiological process that selectively and progressively enhances the cone photoresponse during the first minutes of adaptation to intense light. Because CPTIO is a scavenger of free-radical NO (Akaike et al., 1993; Rand & Li, 1995; Yoshida et al., 1994), this implies that the synthesis and release of NO from retinal cells are increased by intense ambient illumination (Ding & Weinberg, 2007; Eldred, 2001; Neufeld, Shareef, & Pena, 2000) and that CPTIO inhibits the light-responses of cone photoreceptors by removing NO-dependent stimulation of their photoresponses during the early stages of light-adaptation. Burian (1954) was the first to report that the amplitude of the human ERG b-wave increases over time under intense adapting light. In the five decades since then, a similar phenomenon has been observed by many others, in human (Alexander et al., 2006; Peachey, Alexander et al., 1989) as well as other vertebrate ERGs (Bui & Fortune, 2006; Murayama & Sieving, 1992; Peachey, Goto et al., 1993). However, the underlying mechanism of this phenomenon, which might seem at first to contradict the desensitization of cone responses that is expected in light-adaptation (Gouras & MacKay, 1989), is not yet known. The cone membrane potential in the fish retina depolarizes back toward the dark membrane potential under intense adapting light (Burkhardt & Gottesman, 1987). This slow depolarization over time acts to increase the amplitude of the cone photoresponse to additional flashes, in turn causing the amplitude of the cone ERG b-wave also to increase gradually as light-adaptation progresses (Gouras & MacKay, 1989). The results of the present study suggest that NO is released from retinal cells during the initial stages of light-adaptation (Ding & Weinberg, 2007; Eldred, 2001; Neufeld et al., 2000; Sekaran et al., 2005) and that this endogenous NO is a major adaptational regulator of cone photoreceptor activity. Although we found no effect of CPTIO on glutamate-isolated cone ERGs in a previous study (Sato & Ohtsuka, 2010), in that study we did not employ strong light-adaptation, and the condition of the retina was almost fully dark-adapted. Under those conditions, the generation and release of endogenous NO, and therefore also the effect of CPTIO, should have been minimal, as we reported (Sato & Ohtsuka, 2010). From this we may conclude that the release of NO is not involved in the sensitization of rod photoresponses during dark-adaptation. How might NO elicit the effects reported here? Some insight may be derived from recent studies in several model systems, showing that NO can open Ca2+-channels, thereby increasing intercellular Ca2+ concentration and modulating the properties of numerous membrane receptors and channels (Almanza, Navarrete, Vega, & Soto, 2007; Ko & Kelly, 1999; Kourennyi et al., 2004). Such actions require a complete signaling cascade, consisting of: NO source + mediator + effector (NOS – soluble guanylyl cyclase (sGC) – cyclic guanosine monophosphate (cGMP) – cyclic
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Fig. 3. Effect on cone ERGs, of adding different amounts of intravitreal CPTIO to [Glu] = 8 mM. Numbers at the far left correspond to the times when ERGs were sampled, as shown in Fig. 2A (top row). In #1: Control, upward arrow indicates the ERG b-wave amplitude before glutamate + CPTIO injection. Amplitudes of isolated cone ERGs (#3–#5) were averaged 10 before analyzing quantitatively, because they were so small. (A) At lowest dose of CPTIO, isolated cone ERGs (#3–#5) were the same as those after the same amount of glutamate only, as in Fig. 1C. (B) At intermediate dose of CPTIO, cone ERGs (#3–#5) increased less during light-adaptation than in (A). (C) At highest dose of CPTIO, cone ERGs were minimal and amplitudes did not increase during light-adaptation. Time and voltage-calibration bars were the same for (A–C), except in #3–#5, in which the voltage axis is expanded 2.5 (calibration = 0.2 mV).
Fig. 4. Time-course of changes in glutamate-isolated and adaptation-modified cone ERGs, after injection of CPTIO. Filled circles (data from Fig. 2C) represent the control, the ratio of ‘amplitude of cone ERG after application of [Glu] = 8 mM’ to ‘amplitude of ERG b-wave before injection’. The open symbols (data from Fig. 3A–C) represent the same ratio after simultaneous injection of different amounts of CPTIO, shown as the calculated intravitreal concentrations [CPTIO]. The ordinate represents that ratio as percent. Each point and error bar represent the mean ± SD, for n = 5 rats.
nucleotide-gated (CNG) channel), or direct actions of NO, e.g., via Snitrosylation or Tyr-nitration of proteins. Actions of NO on synaptic transmission, which have been well documented in other studies (e.g., Kourennyi et al., 2004), could not have been responsible for the effects that we observed in Glu-treated retinas, in which synaptic transmission from photoreceptors was inactivated. Therefore, we have to seek explanations by mechanisms located in, or closely associated with, the non-synaptic regions of rods and cones. Most authors report that the molecular components of the NOactivated cascade are absent from outer segments, in rat (Kajimura et al., 2003; Kim et al., 1999; López-Costa, Goldstein, & Pecci Saavedra, 1997; Neufeld et al., 2000; Shin et al., 1999; Yamamoto, Bredt, Snyder, & Stone, 1993) as in other species (Blom, Blute, & Eldred, 2009; Haberecht et al., 1998; Koch, Lambrecht, Haberecht, Redburn, & Schmidt, 1994; Neufeld et al., 2000). In any case,
membrane-bound GC, the predominant form and the source of cGMP for transduction in outer segments, is not activated by the NO-donor, sodium nitroprusside (Margulis, Sharma, & Sitaramayya, 1992). Photoreceptor inner segments, however, do contain all the machinery for generating as well as responding to NO – in particular, the NO-synthesizing enzyme, NOS (Haberecht et al., 1998; Kim et al., 1999; Koch et al., 1994; Kurenni et al., 1995; Kurenny et al., 1994; López-Costa et al., 1997; Neufeld et al., 2000; Shin et al., 1999; Yamamoto et al., 1993), and a cGMP target, the cGMP-gated ion channel (Matthews & Watanabe, 1988; Watanabe & Matthews, 1988). The intermediary cGMP-producing enzyme, sGC, a prime NO target, has been detected in inner segments by some investigators (Koch et al., 1994; Nakazawa et al., 2005; Zhang, Beuve, & Townes-Anderson, 2005), but not by others (Kajimura et al., 2003; Blom et al., 2009; Haberecht et al., 1998), perhaps because of problems with antibody cross-reactivity. The rather stout, strongly sGC-immunoreactive rodlike structures in the rat retina, designated outer segments by Nakazawa et al. (2005), may be inner segments, since they appear to arise from the external limiting membrane. The present results are consistent with our previous observation that increasing retinal NO concentration, by intravitreal injection of an exogenous NO-donor (SNAP), suppressed the rod ERG a-wave but enhanced the cone ERG a-wave (Sato & Ohtsuka, 2010). Thus NO is a ‘two-faced’ modulator of retinal function, in that NO released by retinal neurons in response to intense adapting light suppresses the light-evoked activity of (and/or synaptic transmission from) rod photoreceptors, while enhancing the corresponding functions of cone photoreceptors. Such push–pull antagonism not only would provide an ambient-light-dependent mechanism for switching rapidly between rod and cone systems at the input stage to the retina; but under mesopic conditions it could also prevent saturation of inner-retinal signal transmission in those ON and OFF pathways where rod and cone signals converge (Protti et al., 2005). How the cGMP-gated channel and other ion channels regulate cytoplasmic free calcium concentration
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[Ca2+] in inner segments, and how NO might affect rod- and conedependent ERG components in opposite ways, remain unknown, but Zhang et al. (2005) discuss several possibilities. In salamander retina the voltage-dependent calcium-activated currents, IK(Ca) and ICl, were identified electrophysiologically in rod and cone inner segments (Bader, Bertrand, & Schwartz, 1982); a NO-donor, S-nitrosocysteine (SNC), was found to increase a Ca2+-channel current (Kurenny et al., 1994); cGMP enhanced calcium influx into cones, but not into rods (Rieke & Schwartz, 1994); SNC enhanced the depolarization-induced increases of [Ca2+] in rods, but inhibited them in cones (Kourennyi et al., 2004); and NO, working through cGMP and cGMP-gated channels, led to a calcium-induced increase of neurotransmitter release from cones (Savchenko et al., 1997). Plasma-membrane Ca2+-ATPase was reported to extrude Ca2+ more rapidly from cones than from rods (Krizaj, Lai, & Copenhagen, 2003). Since Ca2+ modulates many cytoplasmic signaling cascades and binds to guanylyl cyclase-activating proteins 1 and 2, which are distributed unequally between cones and rods in mammals (Cuenca, Lopez, Howes, & Kolb, 1998), differences in both nucleotide- and Ca2+-mediated signaling may be involved in the differential responses of cones and rods to NO and functional state. Previous studies, utilizing the ERG (Alexander et al., 2006; Bui & Fortune, 2006; Burian, 1954; Gouras & MacKay, 1989; Murayama & Sieving, 1992; Peachey, Alexander et al., 1989; Peachey, Goto et al., 1993; Sato & Ohtsuka, 2010) and single-cell recording (Kourennyi et al., 2004), have shown that NO regulates synaptic transmission from cones to second-order neurons. The present results reveal another, unexpected major role of NO in the retina, which is to regulate (at a stage prior to synaptic transmission) the photoresponses of cones themselves. Finally, differential responses of rods and cones to a wide variety of agents and stimulus conditions appear to be the rule, not the exception, and we have now added nitric oxide as an important differential regulator of light- and dark-adaptation in photoreceptor cells.
5. Conclusion In the present study, we isolated the cone component of the rat ERG (cone photoresponse) by a combination of intravitreal glutamate injection and intense paired-flash stimulation. Under intense adapting light, the amplitude of the isolated cone photoresponse increased gradually, reaching a maximum after about 8 min. This increase was suppressed in a dose-dependent manner by intravitreal injection of the NO scavenger, CPTIO. Our results suggest that NO-synthesis and -release from retinal neurons are enhanced by exposure to relatively intense (photopic) illumination, and that the increase in endogenous NO readjusts cone gain and/or sensitivity during the initial stages of light-adaptation. Since CPTIO is reported to be a specific NO scavenger at the doses used in the present study, it is logical to conclude that NO is the major messenger used by the retina to restore cone signaling rapidly after shifts to higher, and initially saturating, ambient light intensities.
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