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Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts
Submitted 18 March 2004; accepted in final form 2 March 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor agonist kainate and the excitatory amino acid transporter (EAAT) substrate D-aspartate, and both responses were localized to the dendrites. Kainate generated depolarizations whereas D-aspartate had Erev close to ECl and generated hyperpolarizations, indicating that the AMPA/kainate receptors are sign-preserving, whereas the EAATs are sign-inverting. In response to white light, some Cabs gave ON bipolar cell-like responses whereas others gave OFF bipolar cell-like ones, but many cells' responses had both ON and OFF bipolar cell components. In response to appropriately colored center-selective stimuli, many Cabs responded to short and long wavelengths with opposite polarities and were thus double color-opponent. The depolarizing components of the responses to white or colored stimuli were suppressed by the EAAT blocker DL-threo-
-benzyloxyaspartate (TBOA), whereas the hyperpolarizing components were reduced by the AMPA/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX). These results are consistent with the hypothesis that both EAATs and AMPA/kainate receptors are involved in the generation of light-evoked responses in Cabs and that they confer these cells with ON and OFF bipolar cell properties, respectively. Cabs can generate double color-opponent center responses by receiving inputs from certain cones through EAATs and from other cones through AMPA/kainate receptors. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Marc and Sperling 1976; Merbs and Nathans 1992; Tomita et al. 1967). However, the cones themselves cannot discriminate among wavelengths because many combinations of wavelength and intensity can lead to the same response in a cone. Color vision depends on the comparison of signals from two or more cone types by postreceptoral circuits. The neurophysiological basis for color comparison is color opponency, and neurons with this property respond to certain wavelengths with one polarity of voltage change but to others with the opposite polarity.
Substantial color processing takes place within the retina, and thus an understanding of retinal color-opponent mechanisms is crucial for understanding how the brain processes color information. Color-opponent retinal ganglion cells were first reported in goldfish, and subsequent experiments found similar cells in many other species, including primates (Daw 1968a,b; de Monasterio 1978a,b; Marchiafava and Wagner 1981; Wagner et al. 1960). In fish, construction of at least part of the ganglion cells' color-opponent receptive fields begins in the outer retina because many horizontal cells and some bipolar cells are color-opponent (Kaneko 1973; Kaneko and Tachibana 1981, 1983; Svaetichin and MacNichol 1958; Tomita 1965). There are two main types of color-opponent bipolar cells. The single-opponent cells have centers that respond best to long wavelengths and surrounds that respond best to shorter wavelengths or vice versa. On the other hand, double-opponent cells have center and surround responses that change polarity when wavelength changes, e.g., the center depolarizes to long wavelengths but hyperpolarizes to shorter wavelengths, whereas the surround hyperpolarizes to long wavelengths but depolarizes to shorter wavelengths. In fish, most color-opponent bipolar cells are double opponent (Kaneko and Tachibana 1981, 1983; Shimbo et al. 2000). It is generally agreed that horizontal cells mediate the surround responses in bipolar cells, and mechanisms as to how color-opponent surround responses are generated have been proposed (Stell and Lightfoot 1975). However, the mechanisms as to how double-opponent center responses in bipolar cells are generated remain elusive (Shimbo et al. 2000).
In this paper, we investigated the glutamatergic input mechanisms and light-evoked responses of the ON-OFF bipolar cells in the giant danio (Danio aequipinnatus). These cells were morphologically identified by their having axon terminals in both sublaminae a and b of the inner plexiform layer (IPL). Such bistratified bipolar cells have also been identified in two other teleosts, namely goldfish (Sherry and Yazulla 1993) and zebrafish (Connaughton and Nelson 2000; Connaughton et al. 2004). These cells are cone-driven and thus are designated as Cabs (cone-driven bipolar cells with axon terminals in sublaminae a and b) (Sherry and Yazulla 1993), and the present study is the first attempt to characterize their light responses. We will show that many Cabs specialize in color processing, and our findings may shed light on the mechanisms generating center color opponency in fish bipolar cells.
Part of the present communication was previously reported in abstract form (Wong et al. 2003).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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The electrophysiological methods used were similar to those described in Wong et al. (2005b); and all procedures were approved by the Institutional Animal Care and Use Committee at Harvard University. Briefly, dark-adapted adult giant danios (Segrest Farms, Gibsonton, FL; EkkWill Waterlife Resources, Gibsonton, FL; Central Mass Aquatics, Worcester, MA) 1.5–3 in long were anesthetized in tricaine, decapitated, and their eyes removed under dim red light. The retinas were isolated from the pigment epithelium and placed photoreceptor side up on a piece of filter paper (type: 0.45 µm HA; Millipore, Billerica, MA). The retina-filter complex was cut into 200-µm slices using a chopper with Feather blades (Ted Pella, Redding, CA). The slices were incubated in a solution containing 25% (ml/ml) L-15 culture medium (Invitrogen, Carlsbad, CA) and 75% Ringer solution (see following text) with picrotoxin, strychnine, and D-(–)-2-amino-5-phosphonopentanoic acid (D-AP5) omitted at 8°C for 10 min to 8 h prior to recording.
A Dagan 8900 amplifier (Dagan, Minneapolis, MN) was used, and signals were low-pass filtered at 1 kHz for most experiments and at 10 kHz when the capacitative current was used to estimate the series resistance (Rseries). The sampling rates were 2.02 and 20.4 kHz, respectively. After partial compensation, the remaining Rseries was 74.0 ± 3.2 (SE) M
(n = 29). Recordings were made from bipolar cells in the whole cell patch-clamp configuration using glass electrodes with 5- to 10-M tip resistance under continuous superfusion with either control Ringer or drug solutions and under dim red light in the scotopic range. Data were acquired using PCLAMP software (Axon Instruments, Union City, CA).
Chemicals and solutions
The Ringer contained (in mM) 120 NaCl, 2 KCl, 1 MgCl2, 2–3 CaCl2, 4 HEPES, 4 D-glucose, 0.1–0.2 picrotoxin, 0.002–0.005 strychnine, and 0.03 D-AP5, and was set to pH 7.65–7.75 with NaOH. Picrotoxin and strychnine were included to eliminate the influence of amacrine cells on the bipolar cell responses. CaCl2 in the Ringer was substituted with 3 mM CoCl2 in experiments where synaptic release was blocked to ensure direct action of the puffed agonists on the bipolar cells being recorded. The N-methyl-D-aspartate (NMDA) receptor antagonist D-AP5 was also included because D-aspartate, the agonist used to probe for the presence of excitatory amino acid transporters (EAATs) in Cabs, is also an agonist for NMDA receptors (Foster and Fagg 1987).
The intracellular solution contained (in mM) 104 K-gluconate or Cs-methanesulfonate, 12 KCl or CsCl, 0.1 CaCl2, 1 EGTA, 2 Na2-ATP, 4 HEPES, 1 Na-GTP, 0.1–1 Na-cGMP, and 
0.05% (g/ml) Lucifer yellow, and its pH was set to 7.4 with KOH or CsOH. Unless stated otherwise, the cesium-based intracellular solution was used in voltage-clamp experiments, whereas the potassium-based solution was used in current-clamp experiments. Reversal potentials were corrected for Rseries with Ohm's law, Verror = IholdRseries, where Verror is the error in the reversal potential measurement, and Ihold is the holding current at the reversal potential. All membrane potentials were adjusted for the liquid junction potentials, which were 9.033 ± 0.076 mV (n = 6) and 13.43 ± 0.28 mV (n = 6) for cesium- and potassium-based internal solutions, respectively.
Chemicals were either bath-applied or puffed from multi-barrel pipettes (positioned near the dendrites except where noted). L(+)-2-amino-4-phosphonobutyric acid (L-AP4), D-AP5, 6,7-dinitroquinoxaline-2,3-dione (DNQX), and DL-threo--benzyloxyaspartate (TBOA) were purchased from Tocris (Ellisville, MO). Kainate was purchased from Ocean Produce International (Shelburne, Canada). All other chemicals were purchased from Sigma (St. Louis, MO).
Light stimulation
The light source was a 150-W tungsten-halogen bulb. Stimulus flash duration was controlled by an electromechanical shutter. The stimulus was presented through the camera port and focused onto the cell via the water-immersion objective lens. A slit was placed in the light path to restrict the light to an 80-µm-wide slit at the retina. The length of the slit extended from the photoreceptor outer segments to the ganglion cell layer, and the bipolar cell being recorded was positioned at the center of this slit. The white light stimulus had an intensity of 68 µW cm–2 at the retina. Light stimuli of other colors were obtained by inserting narrow-band interference filters into the light path. The wavelengths used and their intensities at the retina were 400 nm (0.26 µW cm–2), 430 nm (2.8 µW cm–2), 460 nm (3.8 µW cm–2), 490 nm (1.8 µW cm–2), 520 nm (14 µW cm–2), 550 nm (10 µW cm–2), 580 nm (4.4 µW cm–2), 610 nm (6.6 µW cm–2), and 640 nm (31 µW cm–2). As the output of tungsten light sources is relatively weak in the ultraviolet (UV) region and the optical components of our setup were not designed to transmit UV wavelengths efficiently, UV stimuli were not tested. Light response data were either single responses or averages of two to six responses.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Cabs were identified by the presence of one or more terminals in each of the two sublaminae of the IPL (Fig. 1, top left). Using the voltage-ramp protocol described in Wong et al. (2005b) and/or the free-running current-clamp recording mode, 1.5–3 mM D-aspartate, 100 µM kainate, and 150–300 µM L-AP4 (agonists for EAATs, AMPA/kainate receptors, and group III metabotropic glutamate receptors, respectively) were puffed onto each of a total of 65 Cabs in the presence of the cobalt-based Ringer. In this Ringer, the mean input resistance of these cells with intracellular cesium was 1.94 ± 0.17 G (n = 27). Of the 65 Cabs recorded, 48 responded to D-aspartate and kainate, 14 to D-aspartate, and 3 only to kainate; L-AP4 never elicited any response. The responses to D-aspartate could be reduced or abolished in the presence of the EAAT blocker TBOA (1–2 mM), confirming that D-aspartate acted specifically and directly on the EAATs of the Cabs recorded (n = 15; not shown).
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Both receptor types were localized to the dendritic region
To investigate further the functions of the EAATs and the AMPA/kainate receptors on Cabs that had both receptor types, we examined their location on these cells. Even though the puffer pipette was placed at the outer plexiform layer (OPL) in the agonist response experiments described in the preceding text, the responses were not necessarily generated in the dendrites as the agonists could have spread to the cell body and axon terminals. To localize the origins of these responses, the puffer pipette was moved to four different positions around the cells, and brief puffs of D-aspartate or kainate applied at each location. For both D-aspartate and kainate, the biggest and fastest responses were obtained when they were puffed onto the OPL (Fig. 2). This suggests that both receptors are mainly on the dendrites. In addition, we encountered eight axonless cells with soma sizes similar to those of Cabs that responded to kainate with Erev close to zero and to D-aspartate with Erev between –60 and –40 mV (not shown), reinforcing the notion that both receptors are on the dendrites. Therefore both receptors might detect glutamate released from photoreceptors and mediate light responses. As these receptors trigger opposite membrane potential changes (Fig. 1, bottom), they would also induce light-evoked responses with opposite polarities. The sign-preserving AMPA/kainate receptors would generate hyperpolarizing light responses, whereas the sign-inverting EAATs would generate depolarizing ones. Evidence that both mediate light-evoked responses is provided in the following text.
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To investigate light-evoked responses, the calcium-based Ringer was used. With internal potassium, the average dark resting potential was –47.4 ± 2.0 mV (n = 24). Center-slit stimuli (see METHODS) were used to minimize stimulation of the surround region of the receptive field. Responses to white light were obtained from 21 Cabs. These responses displayed a variety of waveforms, suggesting the presence of multiple subtypes of Cabs. In addition, the effects of the AMPA/kainate receptor antagonist DNQX and of the EAAT blocker TBOA suggested the participation of both receptor types in the generation of these responses.
Of the 21 Cabs tested with white light, 2 cells responded with mainly OFF bipolar cell-like properties (i.e., hyperpolarization at light on and depolarization at light off, e.g., Fig. 3, cell 1, left), whereas 11 others gave predominantly ON bipolar cell-like responses (i.e., depolarization at light on and hyperpolarization at light off, e.g., Fig. 3, cell 2, left). The responses of the remaining eight cells had both OFF and ON bipolar cell-like components, and two examples are illustrated in Fig. 3 (cells 3 and 4, "control" column). Cell 3 gave a transient depolarization shortly after the beginning of the stimulus, reminiscent of the response of an ON bipolar. However, this transient depolarization was immediately followed by a hyperpolarization during the stimulus, and a transient depolarization was generated at light off, characteristics of the response of an OFF bipolar. Likewise, cell 4 responded to white light with both ON and OFF bipolar cell-like components. It gave a depolarizing response that was sustained throughout the duration of the 1-s stimulus and a transient hyperpolarization at the termination of the stimulus, both characteristics of an ON bipolar cell response. However, a transient depolarization immediately followed the hyperpolarization at light off, similar to the response of an OFF bipolar cell. In short, the responses of Cabs to center-slit white flashes displayed a variety of waveforms, perhaps indicating that multiple physiological subtypes of these bipolar cells exist. An explanation for how these diverse responses may be generated is presented in the DISCUSSION.
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The opposite result was obtained when TBOA was applied. A total of 15 Cabs were tested, including 10 ON bipolar-like cells and 5 hybrid cells. With the EAATs blocked, ON bipolar-like components in the responses were suppressed and replaced by OFF bipolar-like ones in a majority of cases (n = 11); two examples are shown in Fig. 3 (cells 2 and 4). Notice that the dark resting potentials of both cells 2 and 4 became more positive in the presence of TBOA, consistent with a reduction in chloride influx through EAATs. In the remaining four cells tested, all light responses were blocked by TBOA. When DNQX and TBOA were applied simultaneously, all Cab responses were nearly abolished (n = 4; not shown).
Taken together, these results suggest that the light response of most Cabs is mediated by both AMPA/kainate receptors and EAATs with the former generating OFF bipolar cell-like response components and the latter generating ON bipolar cell-like components. Curiously, because the dark resting potential of Cabs (with intracellular potassium) in the calcium-based Ringer was –47.4 mV, whereas the Erev of Cabs' response to D-aspartate measured with internal cesium was –46.0 mV (see preceding text), light-induced deactivation of EAATs was predicted to result in little voltage change or even slight hyperpolarizations. Thus we performed the aforementioned voltage-ramp experiment to determine the Erev of the D-aspartate response using internal potassium instead of cesium and obtained a value of –61.6 ± 1.7 mV (n = 18), which would enable the generation of depolarizing light responses through EAATs. This difference between the reversal potentials measured with the two internal solutions is not unexpected, because efflux of intracellular potassium through EAATs is involved in the transport of glutamate (Seal and Amara 1999).
As mentioned earlier, some Cabs (n = 4 of 15) lost all light responses in the presence of TBOA (but absence of DNQX). This is in agreement with the agonist response analysis presented above, which showed that some cells (n = 14 of 65) possess only EAATs but not AMPA/kainate receptors.
Many Cabs are double color-opponent
A likely function of bipolar cells using both AMPA/kainate receptors and EAATs was revealed when light responses were elicited by center-slit stimuli of various wavelengths. Blue (400–460 nm), green (490–550 nm), and red (580–640 nm) stimuli were presented to a total of 18 Cabs, 10 of which gave color-opponent responses. In all 10 cases, the responses to short wavelengths were dominated by voltage changes of one polarity and the responses to longer wavelengths by voltage changes of the opposite polarity. In other words, they were double opponent. (Responses to UV light were not examined, and therefore the percentage of color-opponent cells could have been even higher.) Two examples are shown in Fig. 4, left. In the control Ringer, cell 1 depolarized to a blue (430 nm) stimulus but hyperpolarized to a green (550 nm) stimulus, whereas cell 2 hyperpolarized to blue (460 nm) light but depolarized to red (640 nm) light. We did not encounter any triphasic responses (i.e., responses of 1 polarity to the shortest and the longest wavelengths and of the opposite polarity to the middle wavelengths) (Shimbo et al. 2000), but again, the exclusion of UV stimuli in our analysis could have caused any triphasic cells to be overlooked.
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Therefore one of the likely functions of using both sign-preserving AMPA/kainate receptors and sign-inverting EAATs to mediate light responses is the generation of double color-opponent receptive field center responses. A Cab may receive inputs from shorter-wavelength cones through AMPA/kainate receptors (or EAATs) to generate hyperpolarizing (or depolarizing) responses to shorter wavelengths but from longer-wavelength cones through EAATs (or AMPA/kainate receptors) to generate depolarizing (or hyperpolarizing) responses to longer wavelengths.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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EAATs in the CNS typically play supportive and modulatory roles, e.g., maintaining extracellular glutamate below neurotoxic levels, re-uptake of glutamate into the presynaptic terminal for the re-cycling of glutamate, reduction of synaptic release through negative feedback, prevention of neurotransmitter spillover, etc. (reviewed in Amara and Fontana 2002; Danbolt 2001). The mediation of light-evoked responses by EAATs on teleost retinal ON bipolar cells was the first demonstration that EAATs can function as glutamate receptors (Grant and Dowling 1995, 1996; Wong et al. 2005a,b). In the present communication, we have provided evidence that EAATs also directly mediate synaptic transmission from cone photoreceptors to another type of teleost retinal bipolar cell, namely the ON-OFF bipolar cells, further illustrating the importance of EAATs at the first synapse of the visual system in teleosts.
It remains to be tested whether the EAATs also play such a role in the OFF bipolar cells. Anatomical evidence for EAATs on OFF bipolar cell dendrites has been reported in various nonteleost species (Eliasof et al. 1998a,b; Euler and Wässle 1995; Fyk-Kolodziej et al. 2003, 2004; Grünert et al. 1994; Rauen and Kanner 1994; Rauen et al. 1996; Reye et al. 2002). For teleosts, two anatomical studies of the distribution of GLT-1a (i.e., EAAT2A) and EAAC1 (i.e., EAAT3) in the goldfish retina showed that neither was expressed in the dendrites of OFF bipolar cells (Schultz and Stell 1996; Vandenbranden et al. 2000). However, the expression of other EAATs in teleost OFF bipolars (or other bipolar cell types, for that matter) has not been examined.
ON-OFF bipolar cells in other species
The present study has provided the first electrophysiological evidence that both sign-preserving and sign-inverting glutamate receptors can co-localize to the same teleost retinal bipolar cell and that both can mediate light responses. Consistent with our findings, electron microscopic studies have shown that some bipolar cells in carp and catfish retinas make both invaginating (presumably sign-inverting) (Stell et al. 1977) and noninvaginating (presumably sign-preserving) (Stell et al. 1977) synapses with photoreceptors (Hidaka et al. 1986; Saito et al. 1985).
In addition to fish, bistratified bipolar cells have been found in turtle and salamander retinas. In turtle, these cells hyperpolarize to white light (Ammermüller and Kolb 1995; Marchiafava and Weiler 1980; Weiler 1981), but in response to colored stimuli, some of them hyperpolarize to certain wavelengths and depolarize to others, i.e., they are color-opponent (Ammermüller et al. 1995). A detailed electron microscopic analysis of one such cell, which depolarized to red and hyperpolarized to green stimuli, revealed that it made invaginating and noninvaginating synapses with red and green cones, respectively, raising the possibility that the color-opponent center responses of these cells are generated by the utilization of different types of glutamate receptors at these synapses (Haverkamp et al. 1999). In another study, Pang et al. (2004) measured light-induced current responses from the bistratified bipolar cells in salamander and showed that these cells had two glutamate receptor mechanisms, one sign-inverting (mediated by metabotropic glutamate receptor type 6, or mGluR6) and one sign-preserving (mediated by AMPA/kainate receptors). Both mechanisms were shown to mediate light-evoked responses, although responses to different wavelengths were not investigated systematically to examine whether these cells were color-opponent. Therefore bistratifying bipolar cells with both sign-inverting and sign-preserving glutamate receptors seem to be common across cold-blooded species, showing that the classical division of bipolar cells into either depolarizing or hyperpolarizing varieties is an oversimplification.
In contrast to our results, Connaughton and Nelson (2000) did not encounter Cabs with both sign-preserving and sign-inverting glutamate receptors in the zebrafish, a species closely related to the giant danio (Meyer et al. 1993). The most likely explanation is that they used the general agonist L-glutamate instead of the EAAT-specific D-aspartate to look for EAATs on the Cabs. As L-glutamate activates not only the EAATs but also the AMPA/kainate receptors, the reversal potentials for the glutamate and the kainate responses might not have differed enough to reveal clearly the presence of EAATs, and the differences were probably made even less obvious by the 20-mV incremental voltage steps used in that study.
Functions of Cabs
Widening the response dynamic range. In response to white light stimulation, Cabs display three main types of response waveforms, and two of these three types are illustrated in Fig. 3. The response of cell 1 resembled that of an OFF bipolar cell, whereas the response of cell 2 was similar to that of an ON bipolar. In the control Ringer (Fig. 3, left), both AMPA/kainate receptors and EAATs were presumably functional, and cell 1 behaved like an OFF bipolar most likely because the AMPA/kainate receptors dominated over the EAATs, whereas cell 2 responded like an ON bipolar because the EAATs dominated. When the AMPA/kainate receptors on cell 1 were blocked with DNQX, only the EAATs remained operational, thereby converting the response to be ON bipolar cell-like. On the other hand, the converse was true when the EAATs on cell 2 were blocked by TBOA (Fig. 3). An obvious question is why both sign-preserving and -inverting glutamate receptors are used to generate these two response types. A plausible explanation is that because AMPA/kainate receptors and EAATs induce voltage changes of opposite polarities, their responses partially oppose each other and thereby prevent the light responses from saturating easily. This results in a wider response dynamic range than could be achieved with either receptor type alone, and this may be the first function of Cabs.
Creation of nonlinear receptive fields.
On the other hand, in the third type of Cab response to white light, both OFF and ON bipolar cell properties are manifested, as exemplified by Fig. 3, cells 3 and 4. In these Cabs, neither glutamate receptor type obscures the response of the other type, perhaps because the AMPA/kainate receptors and the EAATs respond to presynaptic photoreceptors with different kinetics. For example, one of these receptor molecules might be located farther away from the glutamate release sites and thus responds to changes in glutamate concentration more slowly so that the depolarizing and hyperpolarizing components are separated temporally. As these Cabs depolarize at both light on and light off, their light responses show rectification and frequency doubling. Rectification is responsible for generating the nonlinear receptive field of the Y-type ganglion cells originally discovered in the cat retina (Enroth-Cugell and Robson 1966; Hochstein and Shapley 1976). Y-type-like ganglion cells have been reported in teleost retinas (Bilotta and Abramov 1989; Sakai and Naka 1995). Thus part of the nonlinear properties of these teleost ganglion cells may originate at the dendrites of Cabs by mixing ON and OFF bipolar cell properties in the same cell.
Half (n = 4 of 8) of the Cabs demonstrating hybrid ON and OFF bipolar cell light response properties had very transient responses (e.g., Fig. 3, cell 3). In contrast, all of the cone-driven or mixed-input ON bipolar cells examined (which possess only sign-inverting glutamate receptors) were much more sustained (Wong et al. 2005b). An appealing explanation is that in Cabs, the sign-preserving AMPA/kainate receptor response and the sign-inverting EAAT response act to truncate each other, resulting in phasic voltage changes. However, this does not appear to be the case because blocking one of the two glutamate receptors did not cause the remaining response to become more sustained (n = 3 of 3; e.g., Fig. 3 cell 3). We propose that the transient quality of these responses is mainly due to activation and/or inactivation of voltage-gated channels. A variety of voltage-gated potassium and calcium channels are present in Cabs (Connaughton and Maguire 1998).
Generation of color opponency.
Color-opponent retinal bipolar cells in fish were first reported by Kaneko (1973). The underlying synaptic mechanisms were investigated in a more recent study by Shimbo et al. (2000), using carp. Annuli and center spots at various wavelengths were presented, and the reversal potentials for the responses were estimated by injecting depolarizing or hyperpolarizing currents. The authors concluded that the color-opponent responses to the annuli could be explained by the horizontal cell cascade model proposed by Stell and Lightfoot (1975), which postulates that a network of sign-preserving feedforward and sign-inverting feedback interactions between cones and horizontal cells gives rise to color-opponent responses in the horizontal cells. On the other hand, Shimbo et al. found that the responses to center spots could not be explained by this model and thus were most likely generated by cells/mechanisms other than the horizontal cells. The center responses to some wavelengths were found to reverse at relatively positive membrane potentials; the authors proposed this to be an indication of direct inputs from the cones. In contrast, the responses to other wavelengths had a more negative Erev and were proposed to be mediated by inhibitory transmitters released by amacrine cells onto these bipolar cells' axon terminals.
By using a stimulus intended to activate mainly the center region of the giant danio Cabs, we found that just over half (n = 10 of 18) of these cells were color-opponent. Due to technical limitations, wavelengths in the UV region were not tested. In giant danio, there are UV cones with peak sensitivity at 358 nm (Palacios et al. 1996) that could conceivably elicit Cab responses with different polarities than those triggered by the other three cone types. Therefore the actual percentage of color-opponent Cabs in giant danio could be even higher. Due to the inclusion of picrotoxin and strychnine in our Ringer, the involvement of amacrine cells is unlikely, although we cannot rule out that under conditions where GABAergic and glycinergic synaptic transmission is preserved, amacrine cells could participate in the generation of bipolar cell color opponency. With inputs from amacrine cells blocked and influences from the horizontal cell-mediated surround minimized, the photoreceptor-Cab synapse becomes the most likely site where the color-opponent responses we obtained were generated. Because Cabs have both sign-preserving AMPA/kainate receptors and sign-inverting EAATs on their dendrites, and pharmacologically blocking either of these receptor mechanisms abolished color opponency in these cells, we propose that Cabs receive inputs from certain cones through AMPA/kainate receptors to generate hyperpolarizing responses to certain wavelengths and from other cones through EAATs to generate depolarizing responses to other wavelengths. These two mechanisms are consistent with, respectively, the positively reversing and the negatively reversing mechanisms in the color-opponent bipolar cells described by Shimbo et al. 2000. As mentioned earlier, some bipolar cells in turtle may also use both sign-preserving and -inverting glutamate receptors to generate color opponency, although in that case the sign-inverting mechanism is presumably mGluR6 rather than EAATs (Haverkamp et al. 1999).
Glutamatergic input mechanisms generating bipolar cell responses: a summary
In this series of papers, we have shown by electroretinographic and patch-clamp recordings that EAATs, mGluR6, and AMPA/kainate receptors account for most if not all light-evoked responses of retinal bipolar cells in the giant danio. The various responses recorded reflect the types of glutamate receptors present. ON bipolar cells, which depolarize to light, use sign-inverting EAATs and mGluR6 with the cone-driven cells using the former exclusively and the mixed-input cells using both mechanisms. EAATs and mGluR6 may confer cone and rod inputs with different properties, and interaction between these receptors in the mixed-input ON bipolars may underlie rod-cone suppression in these cells (Wong et al. 2005b). OFF bipolar cells, which hyperpolarize to light, use sign-preserving AMPA/kainate receptors (Wong et al. 2005a), although, as mentioned above, whether or not they also use EAATs remains to be tested. ON-OFF bipolar cells, which display a variety of complex (including color-opponent) responses to light, use both sign-inverting EAATs and sign-preserving AMPA/kainate receptors, probably in various ratios and with varying response kinetics (the present paper).
Such a generalization, namely that the glutamate receptors determine the response polarities of ON, OFF, and ON-OFF bipolars, also appears to apply to species other than teleosts (McGillem and Dacheux 2001; Pang et al. 2004; Thoreson and Witkovsky 1999). The only difference between teleosts and other species is that in the latter, only mGluR6 and AMPA/kainate receptors are believed to be used (Thoreson and Witkovsky 1999). However, EAATs are expressed on the dendrites of many bipolar cells in most nonteleost species examined (e.g., salamander: Eliasof et al. 1998a,b; cat: Fyk-Kolodzie et al. 2004; rat: Rauen et al. 1996), and a review of the literature suggests that mGluR6 may not account for all ON bipolar cell responses in many nonteleost species (Wong et al. 2005b). Thus the involvement of EAATs in photoreceptor-bipolar cell signaling in species besides teleosts has not been ruled out.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Present address and address for reprint requests and other correspondence: K. Y. Wong, Dept. of Neuroscience, Brown University, Box 1953, Providence, RI 02912 (E-mail: Kwoon_Wong{at}brown.edu)
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REFERENCES |
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Ammermüller J and Kolb H. The organization of the turtle inner retina. I. ON- and OFF-center pathways. J Comp Neurol 358: 1–34, 1995.[CrossRef][Web of Science][Medline]
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