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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Bipolar cells are the interneurons between sensory receptors and relay neurons of the retina. One function they serve is to form the circuitry for the divergence and convergence of visual information. They also provide a mechanism for signal gain, most notably during detection of low intensity light stimuli. But the principal design feature of bipolar cells may be to form a substrate for feedback inhibition, thereby permitting an intricate regulation of the visual stream of information. This has been most extensively studied in amphibian retina, where feedback is evident at both ends of the bipolar cell: the dendritic and axonal terminals (Wu, 1994). At dendritic terminals, horizontal cells control the responses of bipolar cells through an as yet unidentified transmitter system. Studies have excluded GABA as the transmitter, at least through any conventional synaptic receptor (Hare & Owen, 1996). Interplexiform cells also synapse at the dendritic end of bipolar cells in amphibian retina, where they activate glycine receptors (Maple & Wu, 1998). At the axon terminal, GABAergic amacrine cells control the output of bipolar cells. A picrotoxin-sensitive, bicuculline-insensitive receptor mediates most of this feedback (Lukasiewicz et al. 1994). This pharmacology is the fingerprint of the GABAC receptor. There is evidence that metabotropic GABA receptors may also reduce output at the bipolar cell terminal, but this conclusion is more contentious. While one study found that baclofen suppressed L-type calcium channels and transmitter release in a subpopulation (~20%) of bipolar cells (Maguire et al. 1989), a follow-up study did not confirm this observation (Lukasiewicz & Werblin, 1994). Furthermore, immunohistochemical studies in mammalian retina identified GABAC receptors in bipolar cells, but failed to find GABAB receptors (Koulen et al. 1998).
Another subject of controversy is the question of synaptic and non-synaptic localization of bipolar cell inhibitory receptors. This is particularly true of GABAB receptors because, while agonists have pronounced effects on light responses, antagonists have comparatively small effects. Two possible explanations are: (1) that the simple, light-step stimuli used in most experiments are inappropriate for GABAB receptor activation, or (2) that agonists activate non-synaptic receptors. In contrast, experiments have clearly demonstrated that GABAC receptors are synaptic (Wassle et al. 1998) and that they play an important and well-defined role in the generation of light responses (Lukasiewicz & Werblin, 1994; Zhang et al. 1997a; Dong & Werblin, 1998; Jacobs & Werblin, 1998). However, there remains a question about the types of GABAC receptor present at bipolar cell terminals. In amphibians the evidence suggests that the receptor is blocked by picrotoxin. In rat bipolar cells the GABAC receptor is insensitive to picrotoxin (Feigenspan et al. 1993; Pan & Lipton, 1995). The pharmacology of ferret bipolar cell GABAC receptors is similar to that of amphibians (Lukasiewicz & Wong, 1997). This might signify that rat retina is an anomaly, an exception to the general conclusion that picrotoxin sensitivity defines GABAC receptors. However, a report by Gao et al. (2000) indicates that picrotoxin blocks exogenous GABA activation of amphibian bipolar cell GABAC receptors, but imidazole-4-acetic acid (I4AA) blocks synaptically activated GABAC receptors. This pharmacology conforms with studies in fish and rat retina, where I4AA is a GABAC receptor antagonist (Qian & Dowling, 1993), but conflicts with results in amphibian retina where I4AA acts as a weak agonist in bipolar cells (Lukasiewicz & Shields, 1998). Thus, the pharmacology of GABAergic feedback to bipolar cells is an important but unresolved problem.
In this study, the effect of synaptically activated GABA receptors on the output of bipolar cells was assessed utilizing an approach that stimulated amacrine cells (summarized in Fig. 8). This resulted in an increase in ambient amacrine cell feedback to bipolar cells that was independent of the light stimulus protocol. The objective was to evoke a non-prejudicial activation of feedback synapses, while viewing the output of bipolar cells as measured by synaptic currents in the postsynaptic ganglion cells. The results indicate that synaptic GABAB receptors significantly suppress bipolar cell output. Synaptic GABAC receptors also suppress the output of bipolar cells and two pharmacologically distinguishable pathways mediate this inhibition. Thus, the bipolar cell terminal is under complex GABAergic control, mediated by several ionotropic and metabotropic receptors. The feedback may be differentially activated and depends on the nature of the light stimulus or the level of retinal adaptation.
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METHODS |
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Experiments were performed on tissue slices of retina from the aquatic tiger salamander (Ambystoma tigrinum) in accordance with National Institutes of Health and University Animal Care guidelines. The methodology was described in detail previously (Werblin, 1978; Wu, 1987). In brief, the animal was stunned, decapitated and double pithed, and the eye was enucleated. The retina was separated from the pigment epithelium and positioned, photoreceptor side down, on filter paper. The retina and filter paper were sectioned into 200-300 
m thick slices, which were placed in a perfusion chamber and observed with an upright microscope fitted with differential interference contrast optics. The retina was stimulated by an amber, Stanley LED (Nygaard & Frumkes, 1982). Voltage-clamp and current-clamp recordings were obtained from neurons in the amacrine and ganglion cell layers. All procedures were performed under infrared illumination.
Whole cell recordings were obtained with low resistance electrodes (< 5 M
) filled with pipette solution consisting of (mM): 106 potassium gluconate, 5 NaCl, 2 MgCl2, 5 EGTA and 5 Hepes, buffered to pH 7.4 with KOH. In addition, the solution contained an 'ATP regenerating cocktail' consisting of 4 mM ATP, 20 mM phosphocreatine and 50 units ml-1 creatine phosphokinase. Control amphibian Ringer solution consisted of (mM): 111 NaCl, 2.5 KCl, 1.8 CaCl2, 1 MgCl2, 10 dextrose and 5 Hepes, buffered to pH 7.8 and oxygenated. In all experiments, 5-10 M strychnine was added to the Ringer solution to block glycine currents and 50 M D-AP5 was used to block NMDA receptors.
Ganglion cells were voltage clamped at -70 mV to isolate the EPSCs generated by bipolar cells. To ensure that inhibitory currents did not confound the measurement of EPSCs, effects of exogenous GABA were tested. Although GABA can produce a large conductance change in ganglion cells, it produced a negligible current in cells held at -70 mV. This indicates that cells were held close to the reversal potential for inhibitory signals and that the EPSCs were well isolated.
Electrophysiological data were collected with a List EPC-9 amplifier, HEKA Pulse software and a Macintosh G3 computer and analysed with Igor and Excel software. The analog signals were filtered at 5 kHz but were not leak subtracted. Access resistance was generally 6-12 M and was not compensated. Data are expressed as means ± standard deviation.
Imidazole-4-acetic acid (I4AA), 3-aminopropyl-(methyl)phosphinic acid (APMPA), SR95531 and (1,2,5,6-tetrahydropyridine-4-yl)-methylphosphinic acid (TPMPA) were obtained from Research Biochemicals International (Natick, MA, USA). CGP35348 and CGP55845 were obtained from Novartis (Basle, Switzerland). All other chemicals were obtained from Sigma Chemical Co. (St Louis, MO, USA).
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RESULTS |
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The aim of this study was to evaluate the influence of several forms of amacrine cell GABAergic inhibition on the synaptic signalling between bipolar cells and ganglion cells. The action of amacrine cells was determined by tonically stimulating these neurons pharmacologically while recording from ganglion cells. This stimulation can easily be achieved by applying glutamate agonists. But glutamate agonists known to stimulate amacrine cells also depolarize ganglion cells. Examining ganglion cell light responses under voltage clamp can surmount this drawback. In voltage clamp, a glutamate agonist produces a constant current in ganglion cells that is additive with the EPSC, and therefore easily separated. To determine the importance of GABA receptor subtypes, selective antagonists were utilized (Table 1).
Transient ganglion cells
An example of this is shown in Fig. 1A, where a low concentration of a glutamate analogue, 3 M (RS)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolylpropionic acid) (ATPA), was applied to the retina. The dark trace shows the light-evoked excitatory postsynaptic current (L-EPSC) in a ganglion cell held at -70 mV. The light stimulus was 3 s in duration and the ganglion cell response was a transient excitatory current at light onset and offset. During stimulation with 3 M ATPA, a small inward current was produced due to the direct activation of glutamate receptors on the ganglion cell. The L-EPSC at light onset was greatly reduced (by ~90%), and the OFF L-EPSC was slightly reduced (by ~10%). These L-EPSCs are thought to be generated by bipolar cell input to the ganglion cells. In recordings of light responses from 54 transient ganglion cells, ATPA suppressed 66 ± 24% of the ON response and 10 ± 9% of the OFF response (mean ± standard deviation).
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A, L-EPSC of a ganglion cell before and in presence of the glutamate agonist ATPA (3
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The suppressive effect of ATPA on ganglion cell light responses can be reversed if inhibitory amino acid receptors are blocked, as demonstrated in Fig. 1B. This is a recording from the same cell as that shown in Fig. 1A. Application of 100 M picrotoxin, a blocker of GABAA and GABAC receptors, and 5 M strychnine, a blocker of glycine receptors, in combination increased the amplitude and duration of the ON light response (compare dark traces in Fig. 1A and B). In the presence of these antagonists, ATPA reduced the L-EPSC but the suppression produced by ATPA was mitigated by picrotoxin and strychnine (compare stippled traces in Fig. 1A and B). In 19 cells, ATPA in the presence of picrotoxin and strychnine reduced the ON response by 41 ± 8% and the OFF response by 8 ± 5%. Thus, the effect of ATPA on L-EPSCs in ON-OFF ganglion cells is mediated, at least in part, by inhibitory pathways. Subsequent data show that GABA and glycine antagonists block almost all the suppression produced by ATPA. This indicates that ATPA reduces the excitatory connection between bipolar cells and ganglion cells by activating inhibitory feedback to bipolar cells.
ATPA might suppress the bipolar to ganglion cell synaptic signal by a number of mechanisms, both presynaptic and postsynaptic. However, many possible mechanisms can be excluded. ATPA was chosen because it is a selective kainate receptor agonist (Clarke et al. 1997). Consequently, it is unlikely to interfere directly with light responses of ganglion cells since their synaptic signals are mediated primarily by 3-aminopropyl-(methyl)phosphinic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) receptors (Diamond & Copenhagen, 1993; Lukasiewicz et al. 1997). While ATPA had its most marked effect on the ON EPSCs in ganglion cells, it had only a small suppressive effect on the light responses of ON bipolar cells (Fig. 1C, n = 5). Thus, ATPA does not produce its effect by blocking the light responses of bipolar cells or by saturating the synaptic glutamate receptors in ganglion cells. Considering the action of GABA and glycine antagonists, the most plausible explanation of ATPA's action is that it depolarizes amacrine cells and thereby activates reciprocal synapses that inhibit the bipolar cell terminal.
The effect of ATPA on amacrine cells is illustrated in Fig. 1D. This is a current-clamp recording of a neuron in the amacrine cell layer of the retinal slice and it responded to light with transient ON and OFF EPSPs. ATPA depolarized the cell by approximately 20 mV, close to the peak of the original L-EPSP. ATPA at 3 M produced a similar, strong stimulation of every amacrine cell tested (n = 9). This demonstrates that ATPA will excite amacrine cells, and suggests that it will elicit release of inhibitory transmitter.
Sustained ganglion cells
In contrast to effects on transient ganglion cells, the L-EPSCs in sustained ganglion cells were little changed by ATPA. Figure 2A illustrates the light evoked current and voltage responses of a sustained ON ganglion cell. The current-clamp record shows that ATPA depolarized the ganglion cell and consequently reduced the light response amplitude, although the basic waveforms of the responses were very similar. The voltage-clamp traces from the same neuron show that ATPA produced a small, steady inward current and the peak amplitude, as well as the peak to plateau ratio, of the L-EPSC was slightly reduced. However, these effects were minor compared to the effects of ATPA on transient ganglion cells. Furthermore, blockade of inhibitory transmitters had little effect on the light response or the action of ATPA (Fig. 2B). In this example, picrotoxin, strychnine and CGP55845 were applied to block GABAA and GABAC, glycine, and GABAB receptors, respectively. In the presence of these antagonists, ATPA slightly prolonged the light response, but the peak amplitude was hardly changed. Similar observations were made in eight other sustained ganglion cells. A comparison of Figs 1 and 2 demonstrates that both feedback inhibition and the action of ATPA are distinctly different in sustained and transient pathways.
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A, light evoked EPSCs and EPSPs from a sustained ganglion cell before (left) and during application of 3
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We also compared the effect of ATPA to that of baclofen, which is a GABA agonist that has been reported to suppress sustained ganglion cell responses (Slaughter & Bai, 1989; Ikeda et al. 1990; Müller et al. 1992). In voltage-clamp recordings from sustained ON ganglion cells (e.g. Fig. 2C), ATPA had only a slight effect on the L-EPSC while baclofen produced a more marked suppression of the light response (n = 4). This supports the conclusion that ATPA does not stimulate a GABAergic feedback that suppresses the bipolar input to sustained ganglion cells.
GABA receptors involved in the feedback pathway
Returning to the properties of transient ganglion cell light responses, an attempt was made to identify the inhibitory receptors involved in feedback inhibition. Recent attention has focused on the GABAC receptor, which has been shown to mediate the primary feedback pathway from amacrine to bipolar cells. This has usually been tested using picrotoxin, which blocks both GABAA and GABAC receptors. But (1,2,5,6-tetrahydropyridine-4-yl)-methylphosphinic acid (TPMPA) is a more selective antagonist that isolates the action of the GABAC receptor (Ragozzino et al. 1996). The GABAC component of the ATPA effect was examined using 100 M TPMPA, a concentration that produced a maximal response. Figure 3A shows the light response of a transient ON-OFF ganglion cell under control conditions (dark trace) and after application of 3 M ATPA (stippled trace). Characteristically, ATPA produced a steady inward current and reduced the amplitude of both the ON and the OFF responses, with a larger effect on the former. After recovery from these drugs, most GABA receptors other than the GABAC receptor were blocked (GABAA, glycine and GABAB receptors were blocked by 20 M SR95531, 5 M strychnine and 100 M CGP35348, respectively). Under these conditions, the amplitude of the ON response was slightly reduced, the OFF response was enhanced and both responses were more prolonged (compare dark traces in Fig. 3A and B). In the presence of these antagonists, ATPA induced a steady inward current and reduced the ON and OFF light responses, although the effect of ATPA was reduced in the presence of these antagonists (compare stippled trace in Fig. 3A with trace 2 in B). This indicates that part of the effect of ATPA seen in Fig. 3A was due to stimulation of amacrine cells leading to activation of GABAA, GABAB and/or glycine receptors in bipolar cells. Furthermore, when TPMPA was added to the mixture of other antagonists, the effect of ATPA was almost completely abolished. In five cells, ATPA in the presence of strychnine, SR95531 and CGP35348 suppressed 45 ± 7% of the peak ON response and 9 ± 6% of the peak OFF response. After addition of TPMPA, ATPA had a small effect. Two conclusions emerge from these experiments: (1) a significant portion of the inhibitory amacrine cell feedback to bipolar cells relies on TPMPA-sensitive GABAC receptors; and (2) an additional inhibitory receptor mediates a sizeable portion of the feedback inhibition.
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A, effect of 3
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Addressing the second point, the effect of ATPA was examined after ionotropic inhibitory receptors were suppressed. When the retinal slice was treated with 100 M picrotoxin and 10 M strychnine, blocking GABAA, GABAC and glycine receptors, 3 M ATPA still reduced a significant fraction of both the ON and OFF responses (Fig. 3C). While recording from the same cell, the GABAB antagonist CGP35348 (100 M) was added to the mixture of ionotropic GABA receptor antagonists. This blocked most of the ATPA effect (Fig. 3D). Thus, amacrine cell feedback to bipolar cells activates GABAB as well as GABAC receptors. Similar effects were observed in nine cells: ATPA in the presence of picrotoxin and strychnine reduced the peak ON response by 41 ± 8% and the OFF response by 8 ± 5%, and CGP35448 blocked the effect of ATPA. In another five ganglion cells, CGP35348 (in the presence of strychnine and picrotoxin) reduced but did not fully block the effect of ATPA (see below). These experiments demonstrate that the GABAB receptor mediates a significant portion of the inhibitory feedback to bipolar cells.
Figure 3D shows that picrotoxin, strychnine and CGP35348 blocked almost all of the ATPA-induced suppression of the L-EPSC. In some cells the ATPA effect was not totally blocked by these antagonists. A possible explanation is that GABAC receptors were not fully blocked by picrotoxin. Although GABAC receptors have been shown to be key regulators in the retinas of many species, there is a discrepancy about the pharmacology of these receptors. In several studies on amphibian and mouse retina, picrotoxin completely blocked the GABAC receptor response, while I4AA was ineffective. But Gao et al. (2000) found that GABAC synaptic receptors were blocked by I4AA, not picrotoxin. Both picrotoxin and I4AA are effective antagonists in goldfish horizontal cells (Qian & Dowling, 1994). The ATPA protocol provides a different method of identifying synaptic GABAC receptors since it stimulates amacrine cell synapses that might not be excited by a particular light protocol.
The effects of several GABAC receptor antagonists on the ganglion cell light response were tested while using ATPA to stimulate inhibitory amacrine cells. In the ON-OFF ganglion cell illustrated in Fig. 4A, ATPA suppressed almost all of the ON L-EPSC, but had little effect on the OFF response. After application of an inhibitory cocktail of picrotoxin, strychnine, SR95531 and CGP35348 (PSSC), both the ON and OFF light responses were enhanced and prolonged (Fig. 4B). ATPA was still able to reduce the ON light response, although much less effectively than under control conditions. However, when I4AA was added to the other antagonists, the effect of ATPA was almost completely eliminated. Therefore, I4AA was able to block an additional element of ATPA-induced inhibition. This type of experiment was performed in eight ganglion cells. In four cells, the inhibitory cocktail (PSSC) completely blocked the effect of ATPA, and I4AA had no additional effect. In three cells, the PSSC cocktail did not fully block ATPA's action but the cocktail plus I4AA did. In one cell, I4AA produced an effect beyond that of the cocktail, but ATPA was still able to suppress 18% of the ON L-EPSC. These observations, coupled with the data below, indicate that there is a picrotoxin-insensitive synaptic GABAC pathway in a subset of ganglion cells.
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A, L-EPSC in a ganglion cell before and during application of 3
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The actions of GABAC receptor antagonists on the normal ganglion cell L-EPSCs were examined. For example, in the ON-OFF ganglion cell illustrated in Fig. 5A, glycine, GABAA and GABAB receptors were blocked by a combination of 5 M strychnine, 20 M SR95531 and 100 M CGP55845 (SSC) (dark trace). Addition of 20 M picrotoxin, which inhibited the GABAA and GABAC receptors, enhanced the light response. Increasing the picrotoxin concentration to 100 M did not further alter the L-EPSCs (traces 1 and 2 in Fig. 5A nearly superimpose). This indicates that 20 M picrotoxin produced a saturating drug effect. However, if 20 M I4AA was then added, there was a slight increase in both the amplitude and the duration of the light response, particularly the ON response in this neuron. This points to an I4AA effect on the L-EPSC beyond the maximal effect of picrotoxin.
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A, light-evoked EPSC in the presence of GABAA, GABAB and glycine receptor blockers (20
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We then determined the incremental effect of I4AA on the light response of ON-OFF ganglion cells. Neurons were treated with a broad spectrum of inhibitory amino acid antagonists that block glycine, GABAA, GABAB and GABAC receptors (100 M picrotoxin, 5 M strychnine, 20 M SR95531 and 100 M CGP55845). The L-EPSCs were measured under these conditions, and then again after 20 M I4AA was applied with the other antagonists. Of 11 cells tested, I4AA enhanced the L-EPSCs in five of the cells and had little effect in the other six. Even within the five cells affected by I4AA, the relative effectiveness of picrotoxin and I4AA was variable, probably manifesting a diversity of receptors and cell types within the umbrella of ON-OFF ganglion cells. Figure 5A represents the group of six neurons in which I4AA had little effect. After prior treatment with picrotoxin, I4AA only slightly enhanced the ON L-EPSC but had little effect on the OFF L-EPSC. The neuron shown in Fig. 5B is one of three in which both the amplitude and duration of the ON response were significantly enhanced by I4AA, but the OFF response was little changed. In two other neurons, one shown in Fig. 5C, the enhancement produced by I4AA was very marked both in duration and in amplitude, and I4AA had a larger relative effect on the OFF L-EPSC. Thus, there appears to be a variable relative distribution of picrotoxin-sensitive and picrotoxin-insensitive putative GABAC receptors in retinal ganglion cells.
GABAA receptor action at the bipolar cell synapse was evaluated by blocking GABAB and GABAC receptors, as well as glycine receptors. GABAB receptors were blocked with 100 M CGP55845 and glycine receptors were blocked with 5 M strychnine. GABAC receptors were blocked with either 100 M TPMPA or 150 M APMPA + 20 M I4AA (Fig. 6). In the presence of these antagonists ATPA produced a sustained inward current in the ganglion cell and a small reduction in light response amplitude. When SR95531, a potent antagonist of GABAA receptors, was added to this mixture the effect of ATPA was very slightly less. This indicates, in agreement with Lukasiewicz & Werblin (1994), that GABAA feedback to bipolar cells is a minor contributor to presynaptic regulation at this synapse.
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L-EPSCs were monitored in a ganglion cell in the presence of GABAB, GABAC and glycine receptor blockers. Then 3
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Since GABAA receptors have very little influence on the EPSC, we could evaluate the relative effectiveness of picrotoxin at GABAC receptors without concerns about its action on GABAA receptors. The objective was to determine if TPMPA blocked the same group of synaptic GABAC receptors blocked by picrotoxin, and if I4AA blocked all synaptic GABAC receptors, both picrotoxin sensitive and picrotoxin insensitive. Picrotoxin and TPMPA enhance transient ON and OFF EPSCs in retinas that have been treated with 5 M strychnine, 20 M SR95531 and 100 M CGP55845. But after application of 100 M TPMPA, 100 M picrotoxin produced a further enhancement of ON and OFF EPSCs (Fig. 7A). In contrast, if 100 M picrotoxin was applied first then 100 M TPMPA did not produce an additional effect on the EPSCs (Fig. 7B). This signifies that TPMPA blocks a subset of the picrotoxin-sensitive GABAC receptors. We have shown that I4AA produces an effect beyond that produced by picrotoxin. But the converse is also true. As shown in Fig. 7C, 20 M I4AA enhanced and prolonged ON and OFF EPSCs, but the addition of picrotoxin produced a large additional enhancement. Thus, I4AA does not block all of the picrotoxin-sensitive GABAC receptors.
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A, the L-EPSC was recorded from a ganglion cell in the presence of 100
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DISCUSSION |
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These experiments demonstrate that a multifaceted GABAergic feedback regulates transmitter release from bipolar cell terminals (Fig. 8). This feedback is mediated approximately equally by GABAB and GABAC receptor inhibition, with an additional small GABAA component. The GABAC feedback activates at least two pharmacologically distinguishable GABAC receptors.
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When ATPA was used to stimulate GABAergic amacrine cells, it resulted in inhibition of glutamate release from bipolar cells.
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Our experimental protocol discloses an array of synaptic feedback that can be evoked by ATPA stimulation. But it does not reveal whether these mechanisms normally work in parallel or are activated serially based on parameters of light stimulation. Several studies indicate that both GABAB and GABAC receptor activation truncates the duration of transmitter release, consistent with a delayed feedback model. However, the effects of GABAC antagonists on the light response indicate that these receptors can also suppress the peak of the light response (Fig. 5). This is not surprising since the rise time of transient amacrine cells is fast compared to that of bipolar cells, and thus amacrine cell feedback can alter the early phase of the bipolar cell output.
Metabotropic GABA receptors
There is considerable evidence that GABAB receptor activation can modulate channel activity and alter light responses in ganglion cells in fish, amphibian and mammalian retinas (Bai & Slaughter, 1989; Maguire et al. 1989; Ikeda et al. 1990; Bindokas & Ishida, 1991; Müller et al. 1992; Zhang et al. 1997b). In amphibian retina, there is conflicting evidence on whether GABAB agonists can modulate bipolar cell presynaptic calcium channels, although the agonist baclofen has been shown to suppress synaptic communication between bipolar cells and ganglion cells (Maguire et al. 1989; Slaughter & Bai, 1989; Lukasiewicz et al. 1994). One limitation of these studies has been the use of exogenous agonists and the failure to demonstrate a synaptic activation of GABAB receptors. In this regard, Tian & Slaughter (1994) demonstrated an effect of GABAB receptor antagonists on the light response of ganglion cells and Cook & McReynolds (1998) demonstrated synaptic GABAB receptors linked to a potassium conductance in some ganglion cells. However, the magnitude and extent of these observations are disappointing when compared to the robust effects of GABAB agonists. This has led to debate about the significance of the baclofen-induced effects and questions about whether the effects of agonists are non-synaptic and thus epiphenomenona. The ATPA experiments indicate that synaptic GABAB receptors are important modulators at bipolar cell synapses. This implies that the conventional light step may not be the ideal stimulus to evoke activation of GABAB receptors. One possibility is that a more prolonged stimulus, such as that produced by repetitive stimulation, evokes a greater GABAB receptor activation. This would be consonant with the prolonged amacrine cell stimulation produced by ATPA as well as the metabotropic properties of GABAB receptor transduction. A precedent for this is the auditory system, where GABAB receptors are specifically associated with high frequency stimulation (Brenowitz et al. 1998).
Studies using baclofen have demonstrated that this agonist enhances transient and suppresses sustained ganglion cell responses in amphibian and cat retina (Slaughter & Bai, 1989; Ikeda et al. 1990; Müller et al. 1992). Yet ATPA produced a GABAB receptor-mediated suppression of transient output from bipolar cells, but not of the sustained output. Figure 2 demonstrates that baclofen suppresses sustained responses under the same conditions in which ATPA has no effect. Thus, the effects of baclofen are opposite to those of ATPA. One interpretation is that the effects of baclofen are non-synaptic. However, it is possible that ATPA is not a sufficient stimulus to evoke activation of GABAB receptors in the sustained pathway.
A GABA receptor that was not addressed in this study is the cis-aminocrotonic acid-sensitive receptor that has been found in goldfish bipolar cells and amphibian ganglion cells (Heidelberger & Matthews, 1991; Zhang et al. 1997b). This receptor is also metabotropic but an antagonist has not been developed. Consequently, we could not establish the potential significance of this receptor at the bipolar cell synapse. However, there was a DC component to the suppressive effect of ATPA that we could not eliminate with antagonists. It is possible that this might result from an effect of this other metabotropic GABA receptor.
The ionotropic GABAC receptor
The GABAC receptor suppresses the output of bipolar cells. Our experiments confirm the extensive studies from Lukasiewicz and colleagues (Lukasiewicz & Werblin, 1994; Lukasiewicz, 1996; Lukasiewicz & Wong, 1997; Lukasiewicz & Shields, 1998). However, while most previous studies conclude that picrotoxin suppresses GABAC receptors in bipolar cells, our experiments indicate that there is a picrotoxin-insensitive GABAC receptor that is blocked by I4AA.
We have concluded that I4AA acts by blocking GABAC receptors on bipolar cell terminals. It is unlikely that I4AA is acting on GABAA or GABAB receptors, since these receptors were blocked prior to I4AA application. It is also unlikely that I4AA is acting as a GABAC agonist at the bipolar cell terminal since it enhances and prolongs the EPSCs in ganglion cells. The possibility exists that I4AA acts as a GABAC agonist that inhibits amacrine cell transmitter release, rather than blocking postsynaptic receptors. However, there is little evidence for GABAC receptors in amacrine cells. Furthermore, there is no reason to expect that I4AA would activate amacrine cell GABAC receptors without having an effect on the bipolar cell GABA receptors. There remains the possibility that I4AA has a specific effect on amacrine cell transmitter release that is independent of GABA receptors, but we cannot evaluate this prospect. Based on this rationale and experiments indicating that I4AA is a GABAC antagonist in fish retina (Qian & Dowling, 1994), the most parsimonious interpretation of our results is that I4AA blocks a picrotoxin-insensitive GABAC receptor on bipolar cell terminals.
The presence of a picrotoxin-insensitive, I4AA-sensitive GABAC receptor in amphibian retina is supported by the experiments of Gao et al. (2000), although they concluded that picrotoxin-sensitive GABAC receptors were exclusively non-synaptic and only the I4AA-sensitive receptors were synaptic. This is the opposite of previous conclusions indicating that picrotoxin-sensitive receptors are synaptic and disclaiming the significance of I4AA-sensitive receptors. Our results fall between these extremes and it is likely that we observe both types of synaptic receptor because our amacrine cell stimulation is direct, prolonged, and not predicated on a particular light stimulus protocol. A coalescence of all these studies suggests that there are at least two types of synaptic GABAC receptor and that their relative importance may vary depending on ganglion cell type and ambient conditions that have yet to be ascertained.
Inhibition of visual pathways
GABAergic amacrine cells appeared to preferentially suppress transient input to ganglion cells, largely sparing sustained EPSCs from bipolar cells. Since the stimulation of amacrine cells is independent of light stimulation and is presumably uniform, this indicates that the bipolar cells responsible for the generation of sustained responses are not subject to the same inhibitory regulation. This implies that there are separate sets of bipolar cells responsible for the generation of sustained and transient signals in the inner retina.
This observation does not imply that the sustained pathway is not under inhibitory control. Previous experiments clearly connote the contrary (Frumkes et al. 1981; Miller et al. 1981; Belgum et al. 1984, 1987). However, the evidence indicates that the sustained pathway is regulated primarily by postsynaptic inhibition, while the transient pathway is regulated largely by presynaptic inhibition. A caveat to this conclusion, mentioned above, is that ATPA may not be effective in stimulating the pathway mediating sustained inhibition.
Our previous experiments, performed by exogenous application of GABA, revealed that the ON pathway was more susceptible to GABAergic inhibition, but did not indicate whether synaptic receptors were involved (Zhang & Slaughter, 1995). The present studies clarify that synaptic GABAergic inhibition preferentially suppresses the ON pathway. In the vast majority of ganglion cell recordings in the salamander slice preparation, the suppression of the ON system was larger than that of the OFF system. This is unlikely to be an artifact of the slice preparation because it is so similar to our previous results which were derived from the intact eyecup. The previous study indicated that the GABAC receptor was preferentially located along the ON pathway, and probably at the ON bipolar terminal. The present results indicate that the metabotropic, synaptic GABAergic system is also concentrated in the ON pathway. A parsimonious interpretation would be that both receptor systems were innervated by the same amacrine cell inputs. In this model, it is also appealing to speculate that the GABAC system is designed for rapid suppression of transient signals, while the GABAB system may be involved in feedback related to prolonged stimulation.
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REFERENCES |
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Acknowledgements
This work was supported by NEI grant no. EY05725.
Corresponding author
W. Shen: SUNY, Department of Physiology and Biophysics, 124 Sherman Hall, Buffalo, NY 14214, USA.
Email: wenshen{at}acsu.buffalo.edu
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