I4AA-Sensitive Chloride Current Contributes to the Center Light Responses of Bipolar Cells in the Tiger Salamander Retina

Fan Gao, Bruce R. Maple, and Samuel M. Wu

Cullen Eye Institute, Baylor College of Medicine, Houston, Texas 77030


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Gao, Fan, Bruce R. Maple, and Samuel M. Wu. I4AA-Sensitive Chloride Current Contributes to the Center Light Responses of Bipolar Cells in the Tiger Salamander Retina. J. Neurophysiol. 83: 3473-3482, 2000. Light-evoked currents in depolarizing and hyperpolarizing bipolar cells (DBCs and HBCs) were recorded under voltage-clamp conditions in living retinal slices of the larval tiger salamander. Responses to illumination at the center of the DBCs' and HBCs' receptive fields were mediated by two postsynaptic currents: Delta IC, a glutamate-gated cation current with a reversal potential near 0 mV, and Delta ICl, a chloride current with a reversal potential near -60 mV. In DBCs Delta IC was suppressed by L-2-amino-4-phosphonobutyric acid (L-AP4), and in HBCs it was suppressed by 6,7-dinitroquinoxaline-2,3-dione (DNQX). In both DBCs and HBCs Delta ICl was suppressed by imidazole-4-acetic acid (I4AA), a GABA receptor agonist and GABAC receptor antagonist. In all DBCs and HBCs examined, 10 µM I4AA eliminated Delta ICl and the light-evoked current became predominately mediated by Delta IC. The addition of 20 µM L-AP4 to the DBCs or 50 µM DNQX to HBCs completely abolished Delta IC. Focal application of glutamate at the inner plexiform layer elicited chloride currents in bipolar cells by depolarizing amacrine cells that release GABA at synapses on bipolar cell axon terminals, and such glutamate-induced chloride currents in DBCs and HBCs could be reversibly blocked by 10 µM I4AA. These experiments suggest that the light-evoked, I4AA-sensitive chloride currents (Delta ICl) in DBCs and HBCs are mediated by narrow field GABAergic amacrine cells that activate GABAC receptors on bipolar cell axon terminals. Picrotoxin (200 µM) or (1,2,5,6-tetrahydropyridine-4yl) methyphosphinic acid (TPMPA) (2 other GABAC receptor antagonists) did not block (but enhanced and broadened) the light-evoked Delta ICl, although they decreased the chloride current induced by puff application of GABA or glutamate. The light response of narrow field amacrine cells were not affected by I4AA, but were substantially enhanced and broadened by picrotoxin. These results suggest that there are at least two types of GABAC receptors in bipolar cells: one exhibits stronger I4AA sensitivity than the other, but both can be partially blocked by picrotoxin. The GABA receptors in narrow field amacrine cells are I4AA insensitive and picrotoxin sensitive. The light-evoked Delta ICl in bipolar cells are mediated by the more strongly I4AA-sensitive GABAC receptors. Picrotoxin, although acting as a partial GABAC receptor antagonist in bipolar cells, does not suppress Delta ICl because its presynaptic effects on amacrine cell light responses override its antagonistic postsynaptic actions.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Visual information is segregated into ON and OFF pathways at the outer plexiform layer of the retina, where photoreceptors make glutamatergic synapses on bipolar cells. At photoreceptor synapses on hyperpolarizing bipolar cells (HBCs), glutamate binds to alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors and activates a cation conductance with a reversal potential near 0 mV (Attwell et al. 1987; Maple and Wu 1996; Slaughter and Miller 1983a,b; Wu and Maple 1998). In amphibian retinas, photoreceptor synapses on depolarizing bipolar cells (DBCs) are mediated by L-2-amino-4-phosphonobutyric acid (L-AP4) type glutamate receptors that close a cation conductance with a reversal potential near -10 to 0 mV (Attwell et al. 1987; Maple and Wu 1996; Nawy and Jahr 1990; Slaughter and Miller 1981; Wu and Maple 1998). In fish DBCs, the rod inputs are mediated by L-AP4 receptors, whereas the cone inputs are mediated by a glutamate-activated chloride conductance with a reversal potential near -60 mV (Grant and Dowling 1995; Saito et al. 1979).

In addition to photoreceptors, bipolar cells also receive synaptic inputs from amacrine cells (ACs) in the inner retina (Wong-Riley 1974), and in some species from horizontal cells (HCs) in the outer retina (Naka 1972). These inputs are commonly referred as the lateral or "surround" synapses because HCs and ACs have long lateral processes that convey visual signals to a bipolar cell from cells in surrounding regions. Synapses onto bipolar cells from these lateral processes mediate light responses opposite in sign to those mediated by photoreceptors, thus forming the center-surround antagonistic receptive field (Kaneko 1970; Werblin and Dowling 1969). In some species such as the fish, the receptive fields of retinal HCs and ACs are very large, so their responses to a small spot of light (center light stimulus) are small compared with those elicited by a large light annulus (surround light stimulus) (Kaneko 1971). Consequently, the lateral synapses mediate the "surround" light responses, but contribute little to the "center" light responses in bipolar cells (Kaneko 1970). In other retinas such as the tiger salamander, in addition to interneurons with large receptive fields, there are HCs and ACs with narrow receptive fields, and thus they exhibit large responses to small light spots (Lasansky and Vallerga 1975; Skrzypek and Werblin 1983; Vallerga 1981; Werblin et al. 1988). The center light responses of bipolar cells in these retinas may be mediated not only by the photoreceptor-bipolar cell synapses, but also by lateral synapses from the narrow field HCs and ACs. It is important to elucidate the synaptic mechanisms underlying the center light responses of these bipolar cells.

Most previous studies on bipolar cell light responses were carried out with the intracellular recording techniques from eyecup or isolated whole-mount retinas (Hare and Owen 1996; Thibos and Werblin 1978; Yang and Wu 1997). One major disadvantage of that technique is that it does not allow voltage-clamp measurements of light responses at different membrane potentials to separate ionic current components. In this article, we present a systematic examination of light-evoked currents in DBCs and HBCs under voltage-clamp conditions, and the effects of neurotransmitter agonists and antagonists on various ionic currents evoked by center light stimuli. Additionally, the synaptic pathways mediating light-evoked excitatory and inhibitory inputs to DBCs and HBCs are discussed.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Larval tiger salamanders (Ambystoma tigrinum) purchased from Charles E. Sullivan (Nashville, TN) and KON's Scientific (Germantown, WI) were used in this study. The procedures of dissection and making retinal slices were described in previous publications (Werblin 1978; Wu 1987). For dark-adapted experiments, dissection was done under infrared illumination with a dual-unit Fine-R-Scope (FJW Industry, Mount Prospect, IL) or under dim red light. Oxygenated Ringer solution was introduced continuously to the superfusion chamber, and the control Ringer contained (in mM) 108 NaCl, 2.5 KCl, 1.2 MgCl2, 2 CaCl2, and 5 HEPES (adjusted at pH 7.7). All chemicals were dissolved in control Ringer solution. The retinal slices were viewed with a Zeiss ×40 water immersion objective lens modified for the Hoffman modulation contrast optics (Hoffman Modulation Optics, Greenvale, NY). During the experiment, retinal cells as well as the electrode were clearly observed, and for dark-adapted experiments, a TV monitor connected to an infrared image converter (model 4415; COHU, Palo Alto, CA) attached to the microscope was used.

A photostimulator whose intensity and wavelength could be adjusted by neutral-density filters and interference filters, was used. The light was transmitted to the preparation by way of the epi-illuminator and the objective lens of the microscope, and the spot diameter on the retina was adjusted by a diaphragm in the epi-illuminator. The intensity of light sources was measured with a radiometric detector (United Detector Technology, Santa Monica, CA). The intensity of unattenuated 650 nm light (log I = 0) is 1.25 × 107 photons µm-2 s-1.

Voltage-clamp recordings were made with an Axopatch 200A amplifier connected to a DigiData 1200 interface and pClamp 6.1 software (Axon Instruments, Foster City, CA). Patch electrodes of 5 MOmega tip resistance when filled with internal solution containing (in mM) 118 Cs methanesulfonate, 12 CsCl, 5 EGTA, 0.5 CaCl2, 4 ATP, 0.3 GTP, 10 Tris, and 0.8 Lucifer yellow, adjusted to pH 7.2 with CsOH, were made with Narishige or Kopf patch electrode pullers. The chloride equilibrium potential, ECl, with this internal solution is about -60 mV. Estimates of the liquid junction potential at the tip of the patch electrode prior to seal formation varied from -9.2 to -9.6 mV. For simplicity, we corrected all holding potentials in this paper by 10 mV. Bipolar cells were identified by their morphology when filled with Lucifer yellow [DBCs with axon terminals ramified in sublamina b and HBCs in sublamina a of the inner plexiform layer (IPL)] and their characteristic light responses (Wu and Maple 1998).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Light-evoked currents in DBCs

Figure 1A shows the current records from a voltage-clamped DBC in a dark-adapted tiger salamander retinal slice. Currents were recorded at various holding potentials, and a 500-µm light spot, which covered the bipolar cell receptive field center (Skrzypek and Werblin 1983), was presented to the cell at each holding potential. The peak current response reversed near -20 mV. Since the light response was composed of excitatory and inhibitory components with similar time courses, the two components tended to mask each other. To separate the two components, light-evoked currents were measured at the reversal potentials of the two postsynaptic currents. At -60 mV (ECl, see METHODS), the cell exhibited a sustained inward current (Delta IC) mediated predominantly by cation channels at glutamatergic synapses from photoreceptors (Nawy and Jahr 1990). At 0 mV [near EC, the equilibrium potential for glutamate-gated cation channels (Wu and Maple 1998)], light onset and offset gave rise to transient outward currents (Delta ICl), probably mediated by chloride channels at synapses from amacrine [narrow field amacrine cells exhibit large light responses with similar waveforms when stimulated with small light spots (Vallerga 1981)].



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Fig. 1. Current records from a voltage-clamped depolarizing bipolar cell (DBC) in a dark-adapted tiger salamander retinal slice in normal Ringer (A), in 10 µM imidazole-4-acetic acid (I4AA; B), in 10 µM I4AA + 20 µM L-2-amino-4-phosphonobutyric acid (L-AP4; C), and after normal Ringer wash (D). Currents were recorded at 6 holding potentials (from -100, -60, -40, -20, 0, and +40 mV), and a 0.5-s light step [650 nm, -1 log unit attenuation, 500-µm spot, covering the bipolar cell receptive field center (Skrzypek and Werblin 1983)] was delivered to the cell at each holding potential.

To study the photoreceptor inputs to bipolar cells, pharmacological methods were employed to block light-evoked chloride currents (Delta ICl) mediated by inhibitory synaptic inputs. It has been shown that horizontal cells, amacrine cells, and interplexiform cells in the tiger salamander retina use GABA or glycine as their neurotransmitter (Wu 1991; Yang et al. 1991). GABA responses in bipolar cells are mediated predominately by GABAC receptors (Lukasiewicz 1996; Lukasiewicz et al. 1994), and glycine responses in bipolar cells are strychnine sensitive (Maple and Wu 1998). Among all antagonists tested, we found that 5-20 µM imidazole-4-acetic acid (I4AA) was the most effective in blocking light-evoked chloride currents in bipolar cells. I4AA is a GABAC receptor antagonist (Qian and Dowling 1994), and it blocked (or partially blocked) the GABA-induced chloride currents in bipolar cells (see Figs. 6-8). It also acts as a GABA receptor agonist in salamander bipolar cells (Lukasiewicz and Shields 1998a), and in all bipolar cells in normal Ringer, I4AA increased the chloride conductance by variable amounts (at 0 mV, for example, I4AA induced an outward baseline current of 10-60 pA). Therefore the blockade of light-evoked chloride currents by I4AA presented throughout this paper is likely to be mediated by a mixture of antagonistic and agonistic actions on GABA receptors. Figure 1B shows that in the presence of 10 µM I4AA, Delta ICl disappeared and the light-evoked current became sustained at all voltages and reversed near 0 mV, indicating that it was predominately mediated by Delta IC. The addition of 1-2 µM strychnine (not shown) did not exert further effects on the light-evoked current. Delta IC was completely abolished by subsequent addition of 20 µM L-AP4, a specific glutamate analogue for the DBC glutamate receptors (Nawy and Jahr 1990; Slaughter and Miller 1981), to the bath (Fig. 1C). The light responses recovered after washing with normal Ringer (Fig. 1D), indicating that the disappearance of light responses in I4AA + L-AP4 was not due to cell rundown. This experiment was repeated with 12 other DBCs, and all cells exhibited similar responses to I4AA and L-AP4.

Similar experiments were conducted with the order of I4AA and L-AP4 application reversed. Figure 2 shows the current traces of a DBC with the same voltage clamp protocol and light stimuli as the DBC shown in Fig. 1. In normal Ringer (Fig. 2A), the light-evoked current reversed near -30 mV, indicative of a mixture of Delta IC and Delta ICl. In the presence of 20 µM L-AP4 (Fig. 2B), the peak light-evoked current reversed near -60 mV, suggesting that it was mediated predominately by Delta ICl. With subsequent addition of 10 µM I4AA (Fig. 2C), the light-evoked currents were completely abolished. The light responses recovered after washing with normal Ringer (Fig. 2D), indicating that the disappearance of the light responses in I4AA + L-AP4 was not due to cell rundown. This experiment was repeated on 10 other DBCs, and all cells exhibited similar responses to L-AP4 and I4AA.



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Fig. 2. Current records from a DBC in a dark-adapted tiger salamander retinal slice in normal Ringer (A), in 20 µM L-AP4 (B), in 20 µM L-AP4 + 10 µM I4AA (C), and after normal Ringer wash (D) with the same voltage-clamp protocol and light stimuli as the DBC shown in Fig. 1.

These results suggest that the current responses of DBCs to center illumination (500-µm light spots) are mediated by two current components: a L-AP4-sensitive Delta IC and an I4AA-sensitive Delta ICl.

Light-evoked currents in HBCs

Figure 3A shows the responses of a HBC recorded with the same voltage-clamp protocol and light stimulus as the DBC shown in Fig. 1. The peak current response () did not show a reversal potential, but the brief initial response (open circle ) reversed near 0 mV. At the light offset, a large transient current reversed near 0 mV. Since HBCs also receive both excitatory synaptic inputs from photoreceptors and inhibitory synaptic inputs from amacrine cells, we again measured the light-evoked current at the reversal potentials of the two postsynaptic currents. At -60 mV (ECl), the cell exhibited a sustained outward current (Delta IC) mediated predominantly by photoreceptor inputs (Slaughter and Miller 1983a,b). At 0 mV [near EC, the equilibrium potential for glutamate-gated cation channels (Wu and Maple 1998)] the cell gave rise to an outward current (Delta ICl) during the light step and a transient outward current at light offset, both probably mediated by amacrine cells.



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Fig. 3. Current records from a hyperpolarizing bipolar cell (HBC) in from a dark-adapted tiger salamander retinal slice in normal Ringer (A), in 10 µM I4AA (B), in 10 µM I4AA + 50 µM 6,7-dinitroquinoxaline-2,3-dione (DNQX; C), and after normal Ringer wash (D) with the same voltage-clamp protocol and light stimuli as the DBC shown in Fig. 1. Discrete spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs, small arrows, and sIPSCs, large arrows in A) are present.

In addition to light-evoked currents, discrete spontaneous excitatory postsynaptic currents (sEPSCs) were observed in all HBCs (n = 75). The sEPSCs (small arrows in Fig. 3, A and D), mediated by photoreceptors, reversed near 0 mV, and they were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) (Maple et al. 1994). The sEPSC frequency in darkness was high, and it was substantially reduced by light, consistent with the idea that they are mediated by vesicular release of glutamate from photoreceptors in the dark (Maple et al. 1994; Wu and Maple 1998). In some bipolar cells (both DBCs and HBCs), spontaneous inhibitory currents (sIPSCs, large arrow in Fig. 3A), reversing near -60 mV were observed. These inhibitory currents, thought to represent glycinergic inputs from amacrine cells and interplexiform cells, were always abolished by 1-2 µM strychnine (Maple and Wu 1998).

In the presence of 10 µM I4AA (Fig. 3B), Delta ICl disappeared and the light-evoked current reversed near 0-20 mV, indicating that it was predominately mediated by Delta IC. Both the sustained Delta IC during light-ON and the transient OFF response became smaller, and the frequency of sEPSCs decreased. The addition of 1-2 µM strychnine (not shown) did not exert further effects on the light-evoked currents. With subsequent addition of 50 µM DNQX, a specific AMPA/kainate receptor antagonist known to block glutamate receptors in HBCs, HCs, and amacrine cells (Hensley et al. 1993; Yang and Wu 1991a), Delta IC was completely abolished (Fig. 3C). The light responses recovered after washing with normal Ringer (Fig. 3D), indicating that the disappearance of light responses in I4AA + DNQX was not due to cell rundown. We repeated this experiment on 11 other HBCs, and all cells exhibited similar responses to I4AA and DNQX.

We then reversed the order of I4AA and DNQX application on HBCs. Figure 4 shows responses of a HBC recorded using the same voltage-clamp and light stimulus protocols as for the DBC in Fig. 1. In normal Ringer (Fig. 4A), the light-evoked current did not exhibit an observable reversal potential, and this is consistent with a current generated by a simultaneous conductance decrease (Delta IC) and conductance increase (Delta ICl). In the presence of 50 µM DNQX (Fig. 4B), the light-evoked current almost completely disappeared (12 of 15 HBCs). In 3 of 15 HBCs, about 30% of the light-evoked current persisted in 50 µM DNQX. We then added 10 µM I4AA (Fig. 4C); the light-evoked currents (of all 15 HBCs) were completely abolished. The light responses recovered after washing with normal Ringer (Fig. 4D), indicating that the disappearance of the light responses in I4AA + DNQX was not due to cell rundown.



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Fig. 4. Current records from a HBC in a dark-adapted tiger salamander retinal slice in normal Ringer (A), in 50 µM DNQX (B), in 50 µM DNQX + 10 µM I4AA (C), and after normal Ringer wash (D) with the same voltage-clamp protocol and light stimuli as the DBC shown in Fig. 1.

These results suggest that the current responses of HBCs to center illumination (500-µm light spots) are mediated by two current components: a DNQX-sensitive Delta IC and an I4AA-sensitive Delta ICl. Since DNQX blocks most of the glutamatergic synapses in the salamander retina (Wu and Maple 1998), the DNQX-sensitive Delta IC may be mediated not only by the photoreceptors, but also by other synapses that gate a current with reversal potential near 0 mV.

Effects of picrotoxin, bicuculline, TPMPA, CACA, and I4AA on GABA-induced chloride current and light-induced Delta ICl in bipolar cells

We also examined the effects of picrotoxin, another GABAC receptor antagonist (Lukasiewicz et al. 1994), on light-evoked chloride currents in bipolar cells. Since picrotoxin has been shown to suppress the chloride current elicited by puff application of GABA in salamander bipolar cells (Lukasiewicz et al. 1994; Maple and Wu 1996), we combined experiment protocols of light stimuli and puff application of GABA on the same cells. Figure 5 shows that at 0 mV (near EC), 200 µM picrotoxin greatly suppressed GABA-induced chloride currents in both DBCs (Fig. 5A) and HBCs (Fig. 5B), but failed to block the light-evoked Delta ICl. It instead enhanced and prolonged the Delta ICl (picrotoxin exerted little effects on the light-evoked Delta IC at -60 mV). We observed similar results in seven other DBCs and six other HBCs, and only in one DBC was the light-evoked Delta ICl reduced by picrotoxin. These results confirm the previous findings that chloride currents induced by puff application of GABA on bipolar cells are mediated largely by picrotoxin-sensitive GABAC receptors (Lukasiewicz et al. 1994; Lukasiewicz and Shields 1998b). However, light-evoked Delta ICl appeared to persist in the presence of picrotoxin. We also performed the same experiments testing the effects of (1,2,5,6-tetrahydropyridine-4yl) methyphosphinic acid (TPMPA) [another GABAC receptor antagonist (Lukasiewicz and Shields 1998a) 50-150 µM], cis-4-aminocrotonic acid (CACA) [GABAC receptor agonist (Qian et al. 1998) 100 µM], bicuculline (a GABAA receptor antagonist, 100-200 µM), and strychnine (glycine receptor antagonist, 1-2 µM) on chloride currents elicited by light and puff application of GABA or glycine (results not shown). TPMPA exerted effects similar to those of picrotoxin, and bicuculline had little effect on either the light- or GABA-induced chloride currents. CACA caused a substantial baseline current (at 0 mV, for example, an outward baseline current of 30-100 pA was observed). Its effects on light-evoked Delta ICl in bipolar cells were highly variable, and it appeared to have stronger actions in reducing Delta ICl in DBCs (by 0-60% of the peak response) than in HBCs (by 0-20% of the peak response). Strychnine almost completely suppressed the glycine-induced chloride current [consistent with our previous finding (Maple and Wu 1998)], but exerted little effect on the light-evoked Delta ICl.



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Fig. 5. Effects of 200 µM picrotoxin on light-evoked and GABA-induced chloride currents in DBC (A) and HBC (B). Cells were recorded under voltage-clamp conditions at 0 mV (near EC), and a 0.5-s light step (650 nm, -1, 500-µm spot) and a 200-ms puff of GABA [500 µM in puff pipettes at the inner plexiform layer (IPL)] were delivered.

We next examined the effects of I4AA on GABA-induced chloride current using the same protocol in Fig. 5. Figure 6 show that at 0 mV (near EC), 20 µM I4AA almost completely suppressed the light-evoked Delta ICl (similar to the results shown in Figs. 1 and 3), but partially reduced the GABA-induced chloride currents in both DBCs (Fig. 6A) and HBCs (Fig. 6B). While the effects of I4AA on light-evoked Delta ICl were consistent, the effect on GABA-induced chloride current varied greatly from cell to cell. For the GABA concentration (500 µM in puff pipette) used in Fig. 6, 10-20 µM I4AA reduced the GABA-induced chloride current to 5-85% of their control amplitudes (7 DBCs and 6 HBCs), and in one DBC, 20 µM I4AA enhanced the GABA-induced current by about 10%. In view of the fact that I4AA is a competitive GABA receptor antagonist, we also observed the effects of I4AA on bipolar cell responses to lower concentrations of GABA. Responses to 5 µM GABA in the puff pipette (near the lowest concentrations for which puff responses could be observed) also displayed highly varying sensitivity to I4AA (not shown). In these low concentration experiments, the effects of 10 µM I4AA on GABA-induced chloride currents varied from a complete block to no effect whatsoever. This suggests that multiple types of GABA receptors may be present in different proportions in different types of bipolar cells.



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Fig. 6. Effects of 20 µM I4AA on light-evoked and GABA-induced chloride currents in DBC (A) and HBC (B). Cells were recorded under voltage-clamp conditions at 0 mV (near EC), and a 0.5-s light step (650 nm, -1, 500-µm spot) and a 200-ms puff of GABA (500 µM in puff pipettes at the IPL) were delivered.

To compare the actions of I4AA and picrotoxin on light-evoked and GABA-induced currents on bipolar cells, we combined the experiments in Figs. 5 and 6. Similar to the results shown in Fig. 5, Fig. 7 shows that 200 µM picrotoxin enhanced and prolonged Delta ICl and reduced the GABA-induced chloride current in both DBCs and HBCs. Subsequent addition of 20 µM I4AA substantially reduced both Delta ICl and the GABA-induced current. However, Delta ICl in I4AA + picrotoxin was larger and more prolonged than that in I4AA alone (Fig. 6). We obtained similar results from four other DBCs and three other HBCs. These results suggest that although both I4AA and picrotoxin suppressed the GABA-induced chloride current in bipolar cells, their actions on Delta ICl appeared to oppose each other. It is important to note that Delta ICl is mediated by light responses of interneurons that gate chloride conductance in bipolar cells, and thus it can be altered by augmentation of presynaptic light responses as well as blockade of postsynaptic receptors. We next studied what types of interneurons are likely to mediate Delta ICl and how I4AA and picrotoxin alter light responses of these cells.



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Fig. 7. Effects of 200 µM picrotoxin (2nd traces), 20 µM I4AA + 200 µM picrotoxin (3rd traces) on light-evoked and GABA-induced chloride currents in DBC (A) and HBC (B). Cells were recorded under voltage-clamp conditions at 0 mV (near EC), and a 0.5-s light step (650 nm, -1, 500-µm spot) and a 200-ms puff of GABA (500 µM in puff pipettes at the IPL) were delivered.

I4AA and picrotoxin suppress glutamate-induced synaptic transmission between amacrine cells and bipolar cells

To determine the synaptic sites and interneurons mediating Delta ICl in bipolar cells, we studied the actions of I4AA and picrotoxin on bipolar cells by focal application of neurotransmitters. In a previous study, we found that focal application of glutamate to the IPL induces large chloride currents in DBCs and HBCs (Maple and Wu 1996), presumably by depolarizing GABAergic or glycinergic neurons in the inner retina. It is unlikely that glutamate directly activates chloride channels on bipolar cell axon terminals, because the IPL glutamate response is abolished by substitution of Co2+ for Ca2+ in the bath medium (Maple and Wu 1996). The glutamate response therefore appears to involve Ca2+-dependent release of an inhibitory transmitter in the IPL. In Fig. 8 we show that such glutamate-induced chloride currents in DBCs (Fig. 8A) and HBCs (Fig. 8B) can be reversibly blocked by 5-10 µM I4AA. I4AA (10 µM) completely abolished the glutamate response in all seven DBCs examined and in two of three HBCs studied. In one HBC the glutamate-elicited chloride current was only partially blocked by 10 µM I4AA. This result is consistent with the idea that the light-evoked, I4AA-sensitive chloride currents (Delta ICl) in both DBCs and HBCs are mediated by neurons in the inner retina that release a neurotransmitter that gates chloride channels associated with I4AA-sensitive receptors on bipolar cell axon terminals. We also tested the effects of picrotoxin on glutamate-induced chloride currents in DBCs (Fig. 9A) and HBCs (Fig. 9B) in the presence of 1 µM strychnine. In contrast to its effects on light-evoked Delta ICl, 100 µM picrotoxin substantially suppressed the chloride currents induced by focal application of glutamate at the IPL in both DBCs (n = 4) and HBCs (n = 3), suggesting the existence of picrotoxin-sensitive GABAergic synaptic pathways in these bipolar cells.



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Fig. 8. Effects of I4AA on chloride currents elicited by focal application of glutamate at the IPL in a DBC (A) and a HBC (B). Cells were recorded under voltage-clamp conditions at a holding potential of -10 mV (near EC), and a 100-ms puff of glutamate (200 µM in puff pipettes) was delivered at the IPL near the recorded cell. Current responses before I4AA application (control), in the presence of 5 or 10 µM I4AA (I4AA) and after I4AA wash out (wash) are shown.



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Fig. 9. Effects of picrotoxin on chloride currents elicited by focal application of glutamate at the IPL in the presence of 1 µM strychnine (which blocks glycine-induced chloride current) in a DBC (A) and a HBC (B). The cells were recorded under voltage-clamp conditions at a holding potential of -10 mV (near EC), and a 100-ms puff of glutamate (100 µM in puff pipettes) was delivered at the IPL near the recorded cell. Current responses before picrotoxin application (control), in the presence of 100 µM picrotoxin, and after picrotoxin wash out (wash) are shown.

Light responses of amacrine cells are not significantly altered by I4AA, but they are enhanced and prolonged by picrotoxin

Results from the last section suggest that the I4AA-sensitive Delta ICl in bipolar cells is likely to be mediated by interneurons in the inner retina. Since narrow field amacrine cells are largely GABAergic (Yang and Yazulla 1988) and exhibit large responses to the 500-µm center light spots (Vallerga 1981), they are the primary candidates for the interneurons mediating Delta ICl in bipolar cells. I4AA acts as a GABA receptor agonist [in addition to its antagonistic action on GABAC receptors (Lukasiewicz and Shields 1998b)], it is possible that it suppresses light responses of GABAergic amacrine cells by inhibiting bipolar cell inputs. This may contribute to the I4AA-induced suppression of light-evoked Delta ICl in bipolar cells. We therefore studied effects of I4AA and picrotoxin on light responses of narrow field amacrine cells. Figure 10 shows current responses (under voltage-clamp conditions at -60 mV) of a narrow field amacrine cell (morphology revealed by Lucifer yellow fluorescence, processes spread about 200 µm in IPL, not shown) to a 500-nm light spot in normal Ringer (Fig. 10A), in 20 µM I4AA (Fig. 10B) and in 200 µM picrotoxin (Fig. 10C). I4AA did not significantly alter either the light-evoked current response or the baseline current. On the other hand, 200 µM picrotoxin substantially enhanced and prolonged the light-evoked current, possibly by suppressing GABA-mediated inhibition between amacrine cells (Yang and Yazulla 1988). We obtained similar results from eight other narrow field amacrine cells. These results suggest that the I4AA-induced suppression of Delta ICl in bipolar cells is unlikely to be mediated by suppression of presynaptic light responses in GABAergic amacrine cells. It instead probably resulted from the antagonistic (by receptor blockade) and/or agonistic (by receptor saturation) actions of I4AA on GABA receptors in bipolar cells. The picrotoxin-induced enhancement and prolongation of Delta ICl in bipolar cells (Fig. 5), however, are probably mediated by its presynaptic action on amacrine cell light responses. The larger and more prolonged amacrine cell light responses may override the partial antagonistic action (on GABA receptors in bipolar cells) of picrotoxin and result in larger and more prolonged Delta ICl in bipolar cells.



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Fig. 10. Current responses (under voltage-clamp conditions at -60 mV) of a narrow field amacrine cell (morphology revealed by Lucifer yellow fluorescence, processes spread about 200 µm in IPL, not shown) to a 500-nm light spot (650 nm, -1) in normal Ringer (A), in 20 µM I4AA (B), and in 200 µM picrotoxin (C).


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Bipolar cell responses to center illumination are mediated by two postsynaptic currents

Our results demonstrate that responses of DBCs and HBCs in the tiger salamander retina to illumination at the center of their receptive fields are mediated by two postsynaptic currents: Delta IC, a glutamate-gated cation current with a reversal potential near 0 mV mediated by photoreceptor inputs, and Delta ICl, an I4AA-sensitive chloride current with a reversal potential near ECl (ECl = -60 mV in our experiments) mediated by cells in the inner retina. Delta IC in DBCs is suppressed by L-AP4, and in HBCs is suppressed by DNQX, indicative that the former current is mediated by L-AP4 receptors (Nawy and Jahr 1990; Slaughter and Miller 1981), and the latter current is mediated by AMPA/kainate receptors (Slaughter and Miller 1983a,b).

Under physiological conditions, the dark membrane potential of bipolar cells [ranging from -40 to -60 mV (Lasansky 1992; Yang and Wu 1997)] is close to ECl (Miller and Dacheux 1983), and thus the light-evoked voltage responses are mediated predominately by Delta IC, similar to the situation when bipolar cells are voltage clamped at -60 mV (ECl) in our experiments. Near the dark membrane potential, the light-evoked chloride conductance increase, Delta gCl, in bipolar cells, may elicit little chloride current due to a lack of driving force; nevertheless, it reduces the voltage response amplitudes by shunting the photoreceptor inputs. In the presence of background light the driving force for Delta gCl is substantially increased in the DBCs, which can depolarize to potentials far from ECl. Under this condition Delta ICl may contribute greatly to the light-evoked voltage responses. Therefore Delta ICl activated by amacrine cell inputs on bipolar cell axon terminals may influence bipolar cell responses differently under different adaptational conditions.

Synaptic pathways mediating light-evoked chloride currents (Delta ICl) in bipolar cells

Our data show that in both DBCs and HBCs the light-evoked chloride currents, Delta ICl, can be reversibly blocked by 10 µM I4AA. I4AA is known to be a GABAC receptor antagonist (Qian and Dowling 1994; Tunnicliff 1998) and a partial GABA receptor agonist (Lukasiewicz and Shields 1998a), and therefore the neurons that mediate Delta ICl are likely to be GABAergic [I4AA is also known as an imidazoline receptor agonist (Tunnicliff 1998), but imidazoline receptors have not been localized in the salamander retina]. In the tiger salamander retina, subpopulations of HCs and amacrine cells contain GABA (Wu 1991; Yang et al. 1991), and focal application of GABA in Co2+ Ringer (which blocks synaptic transmission) has shown that GABA receptors are localized on the axon terminals but not the dendrites of bipolar cells (Maple and Wu 1996). In Fig. 8 we show that the chloride current elicited by focal application of glutamate at the IPL is I4AA sensitive. Glutamate delivered at the IPL depolarizes ACs and causes GABA release, leading to activation of I4AA-sensitive GABAC receptors that open chloride channels at bipolar cell axon terminals. We propose that the light-evoked chloride currents, Delta ICl, in DBCs and HBCs are also predominately mediated by GABAergic ACs that activate I4AA-sensitive GABAC receptors at bipolar cell axon terminals.

In Fig. 2 we show that when the photoreceptor-DBC synapses are suppressed by 20 µM L-AP4, the light-evoked current in DBCs is predominately Delta ICl with a reversal potential near ECl (-60 mV). This Delta ICl in L-AP4 can be completely abolished by I4AA, suggesting that it is mediated by GABAergic ACs that activate GABAC receptors on bipolar cell axon terminals. Results from previous reports reveal that 20 µM L-AP4 suppresses the depolarizing responses of DBCs and converts it into a hyperpolarizing response. It does not affect the HBC or HC light responses. Additionally, L-AP4 suppresses the AC responses driven by DBCs but not that driven by HBCs (Yang and Wu 1991b). Based on these results, the Delta ICl in L-AP4 in DBCs (Fig. 2B) is likely to be mediated by GABAergic ACs whose light responses are driven by HBCs. Although we cannot rule out the possibility that HCs may mediate Delta ICl in DBCs (Yang and Wu 1991b), the fact that focal application of glutamate at the outer plexiform layer (which depolarizes HCs) elicits little or no chloride current in bipolar cells (Maple and Wu 1996), and the localization of I4AA-sensitive GABAC receptors in IPL (Fig. 5) suggests that the primary synaptic sites mediating Delta ICl are in the inner retina.

When 50 µM DNQX was applied to HBCs (Fig. 4), the light-evoked currents were abolished in 12 of 15 HBCs. This is because DNQX blocks not only the photoreceptor-HBC synapses (thus suppressing Delta IC in HBCs), but also the photoreceptor-HC and DBC-AC and HBC-AC synapses (Hensley et al. 1993; Yang and Wu 1991a). Consequently, HCs and ACs lose their light responses, and thus Delta ICl in HBCs is suppressed. In three HBCs, about 30-50% of the light response persisted in 50 µM DNQX, and the residual currents were blocked by I4AA. Since the predominate DNQX-resistant second-order retinal neurons are the DBCs, there may be an I4AA-sensitive synaptic pathway between DBCs and these HBCs. A recent report on GABAergic bipolar cells in the salamander retina provides a possible anatomical basis for a direct connection between DBCs and HBCs (Yang 1998), but it is also possible that the I4AA-sensitive pathway involves amacrine cells that are excited via N-methyl-D-aspartate (NMDA) receptors at synapses from DBCs (Maple and Wu, unpublished observations).

Our results suggest that the contribution of glycinergic synapses to the light-evoked Delta ICl in bipolar cells is relatively minor in most bipolar cells, although previous studies have suggested that glycinergic amacrine cells and interplexiform cells can exert significant synaptic inputs to bipolar cells (Maple and Wu 1998). The function of glycinergic synaptic inputs in bipolar cells warrants further investigation.

The surround responses of bipolar cells in isolated salamander retinas are not affected by picrotoxin and strychnine (Hare and Owen 1996), and this may imply that the horizontal cell contribution to the surround response is mediated by picrotoxin- and strychnine-resistent synaptic pathways [feedback or feed-forward synapses (Wu 1992)] in the outer retina. In light of the results presented in this study, however, it is possible that at least part of the surround response is generated by amacrine cell inputs at I4AA-sensitive chloride-mediated synapses on bipolar cell axon terminals. Due to its geometry, the retinal slice preparation used in this study is not well suited to the investigation of classical "surround" inputs. It will be interesting to examine whether, in intact retinas, I4AA suppresses the surround responses of bipolar cells.

Multiple types of GABAC receptors in bipolar cells

Our results are consistent with previous reports that GABA responses in salamander bipolar cells are mediated by GABAC receptors, and probably not by GABAA receptors (due to lack of bicuculline sensitivity) (Lukasiewicz et al. 1994). The large variability we have observed for the effects of I4AA and picrotoxin on bipolar cell GABA responses suggests that multiple types of GABAC receptors may be present in bipolar cells, and that these receptors may occur in different proportions on different types of bipolar cells. It is possible that bipolar cells contain at least two different subtypes of GABAC receptors: one is more strongly I4AA sensitive than the other, but both can be partially blocked by picrotoxin (Lukasiewicz and Shields 1998). Puff application of GABA and glutamate to the IPL favors the activation of both types of GABAC receptors (Figs. 5-8), while the light stimuli primarily elicit transmitter release at synapses using the more strongly I4AA-sensitive GABAC receptors (Figs. 1-6). This is not surprising, since molecular cloning studies have shown that multiple subtypes of GABAC receptors, with varying sensitivity to I4AA and picrotoxin, exist in the retina (Qian et al. 1998). It is worth noting that although I4AA is effective in suppressing light and glutamate-induced chloride currents in bipolar cells, it does not always completely block them. In agreement with a previous report (Lukasiewicz and Shields 1998a), I4AA acts as a GABA receptor agonist (in addition to a GABAC receptor antagonist) in bipolar cells since it caused baseline current changes in almost all DBCs and HBCs in this study. Therefore the suppressive actions of I4AA on the light-, GABA-, or glutamate-induced chloride currents in bipolar cells can be caused either by receptor antagonism and/or receptor saturation. I4AA does not, on the other hand, affect the light-evoked current responses of amacrine cells, suggesting that the I4AA-induced suppression of Delta ICl in bipolar cells is not due to presynaptic inhibition of amacrine cell light responses. Although I4AA did not alter the light response in amacrine cells, it blocked the Delta ICl in bipolar cells. The reason for this discrepancy is not totally clear. A possible explanation is that I4AA enhanced the light responses of most DBCs (see Fig. 1, A and B), but decreased the light responses of most HBCs (see Fig. 3, A and B), and thus the postsynaptic sum (may or may not be linear) of the two changes may cancel each other. Further experiments are needed to clarify this issue.

At the first glance it is somewhat surprising to find that 200 µM picrotoxin did not block, but enhanced and broadened, the light-evoked chloride currents in DBCs and HBCs, since the same or even lower doses of this antagonists decreased bipolar cell responses both to focal application of GABA and to glutamate-elicited synaptic transmission from amacrine cells (Figs. 5 and 7) (Lukasiewicz et al. 1994; Maple and Wu 1998). This apparent discrepancy may be resolved, however, by considering the presynaptic effects of picrotoxin. While the GABAC receptors of bipolar cells are partially picrotoxin sensitive, they are not completely blocked by picrotoxin, at least at the concentration range used in our experiments (100-500 µM). At the same time, the GABA receptors on amacrine cells are also picrotoxin sensitive [conceivably more so, due to a larger contingent of GABAA receptors (Dong and Werblin 1998)]. Our results in Fig. 10 show that picrotoxin results in larger and broader amacrine cell light responses, presumably by disinhibiting the inhibitory GABAergic synapses between amacrine cells. This suggests that the picrotoxin-induced enhancement and broadening of Delta ICl in bipolar cells is probably mediated by presynaptic actions in amacrine cells. Picrotoxin partially blocks the GABAC receptors in bipolar cells, but such antagonistic action is out-weighted by the increase of presynaptic light responses in amacrine cells. Consequently, picrotoxin results in a larger and broader Delta ICl (but not Delta IC at -60 mV) in bipolar cells. In Fig. 7 we show that in the presence of picrotoxin, I4AA only partially blocked Delta ICl in bipolar cells (instead of almost completely blocking Delta ICl in the absence of picrotoxin, Figs. 1-6), suggesting that the enhancement and broadening of amacrine cell light responses also partially overcomes the antagonistic action of I4AA on the GABAC receptors on bipolar cells.


    ACKNOWLEDGMENTS

We thank Prof. X. L. Yang and Dr. Roy Jacoby for reading the manuscript.

This work was supported by National Eye Institute Grants EY-04446 and EY-02520, the Retina Research Foundation (Houston), and the Research to Prevent Blindness, Inc.


    FOOTNOTES

Address for reprint requests: S. M. Wu, Cullen Eye Institute, Baylor College of Medicine, 6565 Fannin St., NC-205, Houston, TX 77030.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 27 July 1999; accepted in final form 2 March 2000.


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