Different effects of low Ca2+ on signal transmission from rods and cones to bipolar cells in carp retina

Brain Research 957 (2002) 136–143 www.elsevier.com / locate / brainres

Research report

Different effects of low Ca 21 on signal transmission from rods and cones to bipolar cells in carp retina Hong-Ping Xu a,b , *, Xiong-Li Yang a,b a b

Institute of Neurobiology, Fudan University, 220 Handan Road, Shanghai 200433, PR China Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, PR China Accepted 15 August 2002

Abstract Modulation of signal transmission from rods, red-sensitive (R-) and green-sensitive (G-) cones to bipolar cells by lowering extracellular Ca 21 was studied in the isolated superfused carp retina using intracellular recording techniques. Low Ca 21 (nominally Ca 21 -free) potentiated light responses of rod dominant ON bipolar cells (rod-ON-BCs). On the other hand, responses of cone dominant ON bipolar cells (cone-ON-BCs) driven by G-cones were dramatically decreased whereas those driven by R-cones were hardly changed in low Ca 21 . Similar effects were observed in scotopic and photopic electroretinographic (ERG) b waves, which reflect the activities of ON-BCs driven by rods and cones, respectively. IBMX (100 mM), an inhibitor of PDE, whose effects mimic those of low Ca 21 on phototransduction, increased responses of both rod-ON-BCs and cone-ON-BCs, suggesting that the distinct effects of low Ca 21 described above are attributable to differential modulation of signal transfer from different types of photoreceptors to BCs. Moreover, scotopic ERG P III responses, reflecting the rod activity, were potentiated both in low Ca 21 and in the presence of IBMX (100 mM). Low Ca 21 causes multiple changes in the outer retina, including increase of glutamate release from the photoreceptor terminal, increase of current and voltage responses of photoreceptors to light, alteration of the synaptic gain from photoreceptors to BCs and modulation of mGluR6 pathway in the rod-ON-BCs. Interplay of these changes may account for differential modulation of R-cone and G-cone driven BC responses, as well as the different effects on rod- and cone-ON-BCs.  2002 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Retina and photoreceptors Keywords: Calcium; Synaptic transmission; Photoreceptor; Bipolar cell

1. Introduction Bipolar cells (BCs) that relay signals from photoreceptors to ganglion cells possess a pivotal position in retinal signal transmission [14]. In mammals rod and cone photoreceptors commonly send inputs to separate populations of BCs [45]. While BCs receive mixed input in fish retina, they may be predominantly driven by either rods (rod-dominant) or cones (cone-dominant) [21,24]. Most of the rod-(dominant) BCs seem to be the ON type, but the cone-(dominant) BCs may be either ON type or OFF type [21,24]. The rod-ON-BCs express a 2-amino-4-phos*Corresponding author. Tel.: 186-21-6564-3719; fax: 186-21-55522876. E-mail address: [email protected] (H.-P. Xu).

phonobutyrate (APB) sensitive glutamate receptor (mGluR6) and their depolarizing responses to light are mediated by the opening of cGMP gated cation channels [12,33,34]. In regard to the cone-BCs, the OFF type expresses ionotropic glutamate receptors (iGluRs) [15,12], whereas the cone driven response of the ON type is generated by glutamate-activated chloride current [9,17,18]. It is well documented that Ca 21 modulates synaptic transmission in the outer plexiform layer, in addition to affecting phototransduction in the outer segment of both rods and cones [10,19,28,38]. Moreover, Ca 21 is also shown to regulate the rod-ON-BC mGluR6 pathway by binding to an intracellular site [32,42]. These effects of low Ca 21 no doubt alter the activity of the retinal secondorder neurons. It was previously demonstrated that signals

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03615-6

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from red-sensitive (R-) and short wavelength-sensitive (S-) cones to L-type horizontal cells were differentially modulated by lowering Ca 21 in the superfusion medium ([Ca 21 ] s ) [51]. In this work we examined the effects of lowering [Ca 21 ] s , and thus, the Ca 21 concentration in the extracellular retinal medium ([Ca 21 ] o ), on carp BCs and found that low Ca 21 differentially modulated light responses of rod- and cone-BCs. We have further explored possible underlying mechanisms.

2. Materials and methods

2.1. Preparations Experiments were performed on the isolated, superfused, flat-mounted retina of adult crucian carp (Carassius auratus), as described previously [50,51]. In brief, the animal was anaesthetized by immersion in a 0.05% solution of tricaine methanesulfonate (MS-222) and the eyeball enucleated. Adequate care was taken to minimize pain and discomfort to animals in accordance with NIH guideline for animal experimentation. The retina, isolated from the eyeball was placed in a perfusion chamber with the photoreceptor side up. The preparation was continuously superfused with oxygenated (bubbled with 95% O 2 and 5% CO 2 ) carp Ringer’s consisting of (in mM) 116 NaCl, 2.4 KCl, 1.2 CaCl 2 , 1.2 MgCl 2 , 1 NaH 2 PO 4 , 28 NaHCO 3 , 10 Glucose, buffered to pH 7.7. All the procedures were performed under dim red illumination.

2.2. Photostimulation, electrical recordings and drug application A photostimulator equipped with two almost identical optical paths was used for producing two coincident 8mm-in-diameter diffuse spots around the electrode tip, which were used as test and background lights, respectively. Light intensities and wavelengths of the two beams were changed by neutral density and interference filters. Intensities of monochromatic lights were measured with a calibrated photodetector (UTD-222, United Detector Technology, Santa Monica, CA, USA), and the unattenuated intensity (log I50) was 2.55310 13 quanta cm 22 s 21 . All light intensities referred in this text are in log units relative to this value. The duration of test flashes was set at 500 ms. For intracellular recordings, glass microelectrodes filled with 4 M potassium acetate were used and they had resistances of 50–100 MV when measured in Ringer’s. Cell penetration was facilitated by briefly (5 ms) overcompensating the preamplifier circuit (MEZ 8201, Nihon Koden Corporation, Tokyo, Japan). Electroretinographic (ERG) responses were recorded between an Ag–AgCl wire electrode in contact with the retina at the photo-

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receptor side and a similar reference electrode in the bottom of the chamber [51], and they were differentially amplified (FZG-81, Shanghai Institute of Physiology, Chinese Academy of Sciences, Shanghai, China) with a band pass of 0.1 Hz to 1 kHz. Both intracellular and ERG responses were displayed on a storage oscilloscope and recorded on a pen recorder (Windograf Recorder 40-847400, Gould Instrument System, Inc. Ohio, USA). Data were presented as means6S.D. in the text and means6S.E.M. in illustrations. Nominally Ca 21 -free solution was made by simply removing CaCl 2 from Ringer’s, without addition of calcium chelators and any other divalent ions to balance the absence of Ca 21 . This solution may still contain 10–20 mM Ca 21 [37]. Other drugs were added to the superfusion medium without substitution. Control and drug-containing perfusates were applied through a peristaltic pump (Rainin Instrument Co. Inc, USA). All chemicals were obtained from Sigma Chemical Company (St. Louis, MO, USA).

2.3. Identification of bipolar cells BCs were identified in reference to previously wellestablished criteria [22–24,40,46]. Rod-ON-BCs and coneON-BCs depolarize to all spectral stimuli with a peak sensitivity at 520 and 620 nm, respectively and the difference of the light sensitivity at 500 nm between these two types of cells is around 2 log units. Moreover, typical responses of a rod-ON-BC to moderate light flashes consist of an initial transient depolarization (lasting about 100 ms), followed by a depolarizing plateau, whereas the depolarizing responses of a cone-ON-BC tend to be much squarer than those of the rod-ON-BC [24].

3. Results

3.1. Low Ca 21 differentially modulates light responses of rod-ON-BCs and cone-ON-BCs Rod-ON-BCs were recorded in preparations which were dark adapted for about 30 min after it was set up in the perfusion chamber. A dim 500-nm test flash (log I5 22.96) was presented at intervals of 10 s. Fig. 1A shows the effects of low Ca 21 (nominally Ca 21 -free Ringer’s) on a rod-ON-BC. Switching from normal Ringer’s to low Ca 21 Ringer’s hyperpolarized the cell by about 14 mV (10.7562.21 mV, n54), which was associated with a significant increase of the light response from 7.5 to 18 mV. The response soon (in 1.5 min) returned to the control level when the preparation was set back to normal Ringer’s. Fig. 1B shows the light responses of this cell to flashes of increasing intensities (log I522.96, 22.15, 20.75, respectively) in a faster time scale, recorded in both normal and low Ca 21 Ringer’s. Note that in normal Ringer’s the light responses of the cell tended to saturate at

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Fig. 1. Potentiation of light responses of a rod-ON-BC by low Ca 21 . (A) The cell was recorded under dark-adapted condition. Lowering [Ca 21 ] o hyperpolarized the cell by 14 mV and potentiated its light responses. A 500-nm test flash (log I522.96) was presented at intervals of 10 s. Light signals are indicated by small square waves on the bottom. (B) Light responses of this cell to 500-nm flashes of three different intensities (log I522.96, 22.15 and 20.75) in both normal Ringer’s (control) and nominally Ca 21 free Ringer’s (low Ca 21 ) are shown in a faster time sacle.

a moderate intensity level (log I522.15) and failed to further increase in amplitudes with a further increase of light intensity. In low Ca 21 Ringer’s all these responses significantly increased in size. For the responses of the rod-ON-BCs to a flash of moderate intensity (log I5 22.15), the relative potentiation was 1.9660.12 (n54, P,0.001, paired Student’s t-test). When a cone-ON-BC was impaled, a 680-nm flash of moderate intensity (log I521.59) was repetitively presented at 1 Hz for about 10 min to release the cone system from the dark suppression [52,53]. Fig. 2 illustrates the

effects of low Ca 21 on a representative cone-ON-BC. Test flashes of 680 nm (log I521.59) and 500 nm (log I52 1.36) were alternately presented at intervals of 3 s and the intensities were chosen so that the light responses of the cell to these two flashes were of comparable amplitudes. Low Ca 21 slightly hyperpolarized the cell by about 3 mV, and dramatically suppressed the response of the cell to the 500-nm flash (from 5.6 to 1.5 mV), but hardly affected the response to the 680-nm flash. These effects of low Ca 21 were abolished when the preparation was returned to normal Ringer’s. Similar results were obtained in five

Fig. 2. Low Ca 21 differentially modulates responses of a cone-ON-BC to 680- and 500-nm flashes. Flashes of 680 nm (log I521.59) and 500 nm (log I521.36) were alternately presented at intervals of 3 s. Low Ca 21 hyperpolarized the cell slightly, which was associated with a dramatic reduction of the response to the 500-nm flash. In contrast, low Ca 21 did not much change the light response to the 680-nm flash. Responses to flashes of 680 and 500 nm recorded at different times are shown in a faster time scale under the continuous trace.

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other cone-ON-BCs. On average, low Ca 21 hyperpolarized the cone-ON-BCs by 6.2562.98 mV and the responses to the flashes of 500 and 680 nm in low Ca 21 were 0.2560.11 and 1.0660.25 of those in normal Ringer’s, respectively (P,0.001 for responses to 500-nm flashes and P.0.05 for responses to 680-nm flashes, paired Student’s t-test).

3.2. Low Ca 21 potentiates scotopic b wave, but suppresses photopic b wave We further studied the effects of low Ca 21 on scotopic and photopic ERG b waves, which are supposed to reflect the activities of rod-ON-BCs and cone-ON-BCs respectively [8,44]. Scotopic ERGs were recorded after the retina was dark adapted for 60 min in the superfusion chamber. Low Ca 21 remarkably increased the scotopic b wave amplitudes in response to 500-nm flashes of two intensities (log I523.06 and 22.15) (Fig. 3A). On average, low Ca 21 caused a 1.8560.21-fold increase (P,0.001, paired Student’s t-test) and a 1.8360.18-fold increase (P,0.001, paired Student’s t-test) in scotopic b waves to 500-nm flashes of the two intensities, respectively (n58). Phototpic ERGs were recorded when a background light (500 nm, log I522.15), which suppresses rod light responses completely but with little effects on cones, was presented for at least 15 min and the b waves reached a steady state [51]. In normal Ringer’s, the photopic ERG consists of an initial cornea-negative a wave, a positive b wave and a positive d wave (off response) at the flash offset [14]. The effects of low Ca 21 on photopic ERGs were clearly different from those on scotopic ERGs. Switching to low Ca 21 Ringer’s greatly altered the photopic ERG waveforms. For the photopic ERGs induced by 500-nm flashes of two intensities (log I521.78 and

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20.75), low Ca 21 entirely abolished the b waves, leaving a big negative component and a conspicuous off response (Fig. 3B, left side). Changes in photopic ERGs induced by 680-nm flashes of two intensities (log I521.59 and 20.36) were quite different and characteristic. First, the initial negative a waves were dramatically increased in amplitudes in low Ca 21 (Fig. 3B, right side). Second, the time to peak of the b waves in low Ca 21 was much prolonged, indicating that the b waves appeared with much delay, compared to control, and the b waves (or ERG P II component, which is mainly responsible for b wave) in low Ca 21 were actually generated when the P III components (a waves) had almost returned to baseline. When the b waves recorded in low Ca 21 were measured from the baseline, the amplitudes were not greatly changed, compared to control. Similar results were observed in nine other light-adapted retinas.

3.3. IBMX potentiates responses of both rod- and coneON-bipolar cells Lowering [Ca 21 ] o modulates both phototransduction in the outer segment of photoreceptors and glutamate release from the photoreceptor terminal [51,54]. Therefore, any changes in the activity of BCs caused by low Ca 21 should be a consequence of the above dual action on photoreceptors. To further explore possible mechanisms which underlie the different changes in rod- and cone-ON-BC activities observed, effects of IBMX (3-isobutyl-1methylxanthine) were examined. As an inhibitor of PDE, IBMX exerts effect on phototransduction in the outer segment, similar to that of low Ca 21 , but does not directly affect glutamate release from the photoreceptor terminal, as caused by lowering [Ca 21 ] o . As shown in Fig. 4A,

Fig. 3. Effects of low Ca 21 on ERGs. (A) Low Ca 21 greatly potentiated scotopic b waves induced by 500-nm flashes of two intensities (log I523.06, 22.15). (B) Changes of photopic ERGs in low Ca 21 . ERGs induced by flashes of 500 and 680 nm were recorded in the presence of a background light (500 nm, log I522.15). Lowering [Ca 21 ] o greatly enhanced the a waves and completely suppressed the b waves in response to 500-nm flashes. The a waves elicited by 680-nm flashes were greatly potentiated in low Ca 21 as well, but the b waves persisted. Note that the time to peak of the b waves was remarkably prolonged in low Ca 21 .

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Fig. 4. Effects of IBMX on rod- (A) and cone-ON-BCs (B). IBMX (100 mM) hyperpolarized both the cells and enhanced their light responses. For the rod-ON-BC, 500-nm flash (log I522.96) was presented at intervals of 10 s. For the cone-ON-BC, flashes of 680 nm (log I521.59) and 500 nm (log I521.36) were alternately presented at intervals of 3 s.

application of 100 mM IBMX hyperpolarized the rod-ONBC by 9.5 mV and potentiated its light response to 500-nm flash (log I522.96) from 10 to 16.2 mV, with an average relative potentiation of 1.7660.11 (P,0.001, paired Student’s t-test, n54). IBMX of 100 mM hyperpolarized the cone-ON-BC as well (9.4 mV), but potentiated the light responses of the cell to both 500 nm (log I521.36) and 680 nm (log I521.59) flashes (Fig. 4B), which was different from the effect of low Ca 21 . The relative potentiation obtained from five cone-ON-BCs was 1.7060.23 (P,0.001, paired Student’s t-test) and 1.6960.24 (P,0.001, paired Student’s t-test) for responses to 500- and 680-nm flashes, respectively. It is noteworthy that the responses to the 500-nm flash exhibited waveform changes following the application of IBMX and they were characterized by an initial on-transient component.

test, n57) in size in low Ca 21 and in the presence of 100 mM IBMX, respectively (Fig. 5).

3.5. Discussion In the present work we reported that low Ca 21 potentiated light responses of the rod-ON-BCs in carp retina. In accord with this, the scotopic b wave was significantly increased in amplitude in low Ca 21 . On the other hand,

3.4. Low Ca 21 and IBMX potentiate both scotopic and photopic P III responses Modulation by low Ca 21 and IBMX of photopic ERG P III responses in carp retina was previously examined [51]. The effects of low Ca 21 and IBMX on scotopic P III responses, which are believed to reflect the activity of rods [16,31,48], were further explored in the present work. Scotopic P III responses were obtained by perfusing the retina with 3 mM glutamate containing Ringer’s under dark-adapted conditions. The scotopic PIII response to 500-nm test flash (log I522.96) exhibited a 2.7660.61fold increase (P,0.001, paired Student’s t-test, n58) and a 2.1660.37-fold increase (P,0.001, paired Student’s t-

Fig. 5. Low Ca 21 and IBMX potentiated scotopic P III responses. Data obtained in low Ca 21 and in the presence of 100 mM IBMX are presented as multiples of control. Error bars 6S.E.M. Waveforms shown in the inset are representative scotopic P III responses to 500-nm flash (log I522.96) in normal (control), low Ca 21 or IBMX containing Ringer’s.

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changes in light responses of the carp cone-ON-BCs caused by low Ca 21 were wavelength-dependent. Whereas the responses to 500-nm flashes were dramatically suppressed, those to 680-nm flashes were hardly changed in amplitudes.

3.6. Multiple effects of low Ca 21 in the outer retina It is known that lowering [Ca 21 ] o may produce profound changes in the outer retina, leading to changes in the activity of BCs. First, low Ca 21 causes a reduction of the PDE activity and an increase of the guanylate cyclase (GC) activity in the photoreceptor outer segment [25–28], which makes more cGMP-gated channels open in the outer segment, resulting in a more depolarizing membrane potential of photoreceptors. This has been demonstrated in salamander rods and cones [50]. Depolarization of photoreceptors facilitates voltage-dependent glutamate release from the terminal. In addition, lowering [Ca 21 ] o causes changes of Ca 21 influx into photoreceptors and therefore alter calcium-dependent glutamate release. It was previously thought that low Ca 21 should reduce synaptic release of glutamate, thus causing the ON-BC membrane potential to depolarize, which is just opposite to what we reported in this paper. However, several lines of recent evidence have strongly suggested that lowering [Ca 21 ] o could actually increase, but not decrease, the Ca 21 influx into photoreceptors [36]. In the turtle retina, for instance, it was reported that lowering [Ca 21 ] o induced a leftshift of the I–V curve of Ca 21 currents of both rods and cones, thereby resulting in an increased Ca 21 influx into the photoreceptor terminal [13,35,37]. Moreover, using patch clamp recording and calcium image techniques, Baldridge et al. demonstrated that mild depolarization resulted in an increased intracellular Ca 21 , which was larger in 1 mM [Ca 21 ] o , rather than 3 or 10 mM [Ca 21 ] o [5]. These authors suggest that the above paradoxical effects could be accounted for by modification of surface charges of the photoreceptor membrane. Although there is no direct evidence about an increase of glutamate concentration in the synaptic cleft between photoreceptors and the second-order neurons, all the evidence now available support that lowering [Ca 21 ] o may potentiate glutamate release from the photoreceptor terminal. The increased glutamate release may result in ‘saturation suppression’ of BC light responses, as discussed in a previous work concerning the effects of low Ca 21 on light responses of the carp cone horizontal cell [51]. It was suggested that with an almost saturated background glutamate concentration at the synaptic cleft in low Ca 21 , a definite reduction in glutamate release caused by a light flash may induce a smaller current change of horizontal cells than that recorded when the background level of glutamate is relatively low (as it may occur in normal Ringer’s). Since ON-BC dendrites and horizontal cell processes are closely juxtaposed in the invaginations

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of cone terminals [20], this argument seems to be applicable for the case of BCs. A second action of low Ca 21 is to increase current and voltage responses of photoreceptors to light [7,30,54]. In the carp retina it was previously demonstrated that the photopic P III response was greatly increased in size in low Ca 21 [51]. In the present work it was further shown that the scotopic P III response was remarkably potentiated in either low Ca 21 or in the presence of IBMX as well. The increased voltage responses of photoreceptors in low Ca 21 would definitely enhance BC voltage responses if the synaptic gain were not changed. The above two effects are in opposite signs. Therefore, whether light responses of the second-order neurons are potentiated or suppressed basically depends upon how much the saturation suppression is compensated by the increased voltage responses of photoreceptors.

3.7. Mechanisms underlying the different effects of low Ca 21 on rod- and cone-ON-BCs In the toleost retina, rod- and cone-driven BC light responses are generated by two distinct mechanisms. Whereas the cone-driven response is mediated by the glutamate-activated chloride current [17,18], the rod-driven one is generated by activation of metabotropic glutamate receptors (mGluR6) [12,33,34]. It was recently reported that external Ca 21 appears to inhibit rod-ON-BC cation channel function by regulating the mGluR6 pathway, leading to a desensitization of ON-BC responses [32,42]. This desensitization is attenuated by buffering Ca 21 with BAPTA, a calcium chelator, or removing Ca 21 from the bathing solution [32,42]. Therefore, such a regulation of the mGluR6 pathway by Ca 21 may partly account for potentiated light responses of rod-ON-BCs. There are other factors that could change voltage responses of the retinal second-order neurons. That is the voltage-dependence of synaptic gains in the outer plexiform layer. It is well established that in the physiological range, synaptic gains between rods and the second-order neurons increase with the increased depolarization of the rod membrane potential [2,3,49]. The voltage-dependent Ca 21 channels in the rod terminal are open when rods are depolarized from the dark membrane potential (¯245 mV) and an e-fold increase occurs with each 6 mV of depolarization. This activation property may mainly account for the non-linearity of the rod output synaptic gain [3,4]. In low Ca 21 rods are depolarized by about 7 mV in the salamander retina [54], which makes the Ca 21 channels in the rod terminal working in a higher gain range, thus leading to larger voltage responses of BCs. It is recently found, on the other hand, the synaptic gain from cones to the second-order neurons (horizontal cells) is almost independent of cone polarization when feedback from horizontal cells to cones is active [29]. It is also suggested

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that the linearity of voltage gain is hold between cones and BCs [55]. This difference in voltage-dependent synaptic gains may be also responsible for differential modulation by low Ca 21 of rod- and cone-driven light responses of BCs. The different effects of low Ca 21 on rod-ON-BCs and cone-ON-BCs may be also due to different Ca 21 channel subtypes expressed in the rod and cone terminals. Glutamate release from the rod terminal is prominently mediated by L-type Ca 21 channels [4,11,39,49], whereas in cones, in addition to L-type Ca 21 channels [6], other dihydropyridine insensitive channels [47], such as cGMP-gated cation channels, are involved in neurotransmitter release from the cone terminal [38,41]. Therefore, lowering [Ca 21 ] o might differentially regulate the Ca 21 currents in the rod and cone terminals. Actually, differential modulation of the Ca 21 currents of rods and cones by dopamine and somatostatin have been reported [1,43].

3.8. Possible explanation for differential modulation by low Ca 21 of signals from R-cones and G-cones to BCs It was previously reported that low Ca 21 suppressed the G-cone-driven responses of the cone horizontal cell more substantially than the R-cone-driven ones. This observation was accounted for by that the reduction in the synaptic strength between R-cones and cone horizontal cells due to saturation suppression could be compensated to a larger extent by the more potentiated R-cone signal in low Ca 21 , as compared to the G-cone signal [51]. This difference in compensation extent could be also in fact responsible for differential modulation of R-cone- and G-cone-driven BC responses (Figs. 1 and 2). However, we have noticed that the situation was more complicated at the BC level. While R-cone-driven responses of the cone horizontal cells are suppressed by low Ca 21 , though to less extent, those of the BCs are almost unchanged (Fig. 2). Furthermore, it should be noted that one would think that the b wave elicited by red light in low Ca 21 is much enhanced, if the amplitude were measured from the trough of the a wave to the cornea-positive peak. When it was done, the ERG data may be thought to be inconsistent with that obtained from the BCs. All these remain so far not be well explained. It is reasonable to suggest that other mechanism(s) may be involved into the different effects on BC responses driven by R- and G-cones, respectively.

Acknowledgements This work was supported by grants from the National Program of Basic Research sponsored by the Ministry of Science and Technology of China (G 1999054000), the Shanghai Commission of Science and Technology, Shanghai Institutes of Biological Sciences, Chinese Academy of

Sciences and the Shanghai-Unilever Research & Development Fund (No. 2004).

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