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Published in final edited form as: Vision Res. 2012 Jul 27;68:48–58. doi: 10.1016/j.visres.2012.07.012

Ionotropic glutamate receptors mediate OFF responses in light-adapted ON bipolar cells

Ji-Jie Pang 1, Fan Gao 1, Samuel M Wu 1
PMCID: PMC3733217  NIHMSID: NIHMS495624  PMID: 22842089

Abstract

Previous studies have suggested that photoreceptor synaptic inputs to depolarizing bipolar cells (DBCs or ON bipolar cells) are mediated by mGluR6 receptors and those to hyperpolarizing bipolar cells (HBCs or OFF bipolar cells) are mediated by AMPA/kainate receptors. Here we show that in addition to mGluR6 receptors which mediate the sign-inverting, depolarizing light responses, subpopulations of cone-dominated and rod/cone mixed DBCs use GluR4 AMPA receptors to generate a transient sign-preserving OFF response under light adapted conditions. These AMPA receptors are located at the basal junctions postsynaptic to rods and they are silent under dark-adapted conditions, as tonic glutamate release in darkness desensitizes these receptors. Light adaptation enhances rod-cone coupling and thus allows cone photocurrents with an abrupt OFF depolarization to enter the rods. The abrupt rod depolarization triggers glutamate activation of unoccupied AMPA receptors, resulting in a transient OFF response in DBCs. It has been widely accepted that the DNQX-sensitive, OFF transient responses in retinal amacrine cells and ganglion cells are mediated exclusively by HBCs. Our results suggests that this view needs revision as AMPA receptors in subpopulations of DBCs are likely to significantly contribute to the DNQX-sensitive OFF transient responses in light-adapted third- and higher-order visual neurons.

Keywords: Depolarizing bipolar cells; transient OFF responses; GluR4 receptors; mGluR6 receptors; 6,7-dinitroquinoxaline-2,3-dione (DNQX); cyclothiazide; dark and light adaptation

1. Introduction

The ON and OFF signaling pathways are two primary parallel information channels in the visual system (Dowling, 1987;Rodieck, 1998). Many visual neurons, including third-order retinal cells, cells in the lateral geniculate nucleus (LGN) and the visual cortex, exhibit a depolarizing response or spike increase (ON cells), hyperpolarizing response or spike decrease (OFF cells) to a light step, or a transient depolarization or spike increase at the onset and offset of a light step (ON-OFF cells) (Kuffler, 1953;Werblin & Dowling, 1969;Hubel & Wiesel, 1977). In addition to encoding durations of light stimuli, the ON-OFF cells also register motion and time-varying information in the visual world (Taylor et al., 2000;Roska & Werblin, 2003). For many years, the widely-accepted view has been that ON responses (ON cells and the onset response of ON-OFF cells) are mediated exclusively by the depolarizing bipolar cells (DBCs) and the OFF responses (OFF cells and the offset response of ON-OFF cells) are mediated exclusively by the hyperpolarizing bipolar cells (HBCs) (Hensley et al., 1993;Miller, 1979). The main support for this view is that ON responses in the visual pathway (from third-order retinal cells to cortical neurons) can be selectively abolished by L-AP4 (Schiller et al., 1986;Hensley et al., 1993), a specific metabotropic glutamate receptor (mGluR6) agonist that suppresses DBC responses without affecting the HBC responses (Slaughter & Miller, 1981;Nawy & Jahr, 1991). It has also been shown that AMPA/Kainate receptor antagonists, such as DNQX, CNQX and NBQX, suppress HBC light responses without affecting the DBC light responses (Wu & Maple, 1998;Sasaki & Kaneko, 1996). This explains why the OFF responses of third-order retinal cells are DNQX-sensitive (Massey & Miller, 1988). However, because AMPA/Kainate receptors also mediate synaptic transmission from both DBCs and HBCs to third-order retinal neurons (Hensley et al., 1993;Lukasiewicz et al., 1997;Mittman et al., 1990), their specific blockers cannot be used to selectively block the OFF channels beyond the HBCs, in the same way as L-AP4 can for the ON-channels.

Glutamatergic photoreceptor inputs to DBCs in the vertebrate retina are mediated by mGluR6 receptors (Nawy & Jahr, 1991), with the possible exception of a glutamate transporter-regulated chloride mechanism in some fish DBCs (Grant & Dowling, 1995). The binding of glutamate with mGluR6 receptors closes cation channels in darkness, and light suppresses glutamate release from photoreceptors, opens cation channels and depolarizes the DBCs (Shiells & Falk, 1990). There has been sparse evidence suggesting AMPA/Kainate receptors exist in mammalian DBCs (Kamphuis et al., 2003), but their function is unknown. In this article, we present evidence showing that in addition to the well-known mGluR6 receptors that mediate the DBC’s sign-inverting, depolarizing light responses, certain types of DBCs use AMPA receptors to generate a transient, sign-preserving OFF response under light-adapted conditions. Our results also suggest that the DNQX-sensitive transient OFF responses in higher-order visual neurons under light adapted conditions are not generated exclusively by the HBCs.

2. Methods

Larval tiger salamanders (Ambystoma tigrinum) purchased from Charles D. Sullivan, Co. (Nashville, TN) and KON’s Scientific Co. Inc. (Germantown, WI) were used in this study. All animals were handled in accordance with the policies on treatment of laboratory animals of Baylor College of Medicine and the National Institutes of Health. Before each experiment, salamanders were anesthetized in MS222 (2gm/liter) until the animal gave no visible response to touch or water vibration. The procedures of dissection, retinal slicing and recording were described in previous publications (Wu, 1987b;Pang et al., 2008). Dissection and recording were done under infrared illumination with a dual Nitemare infrared scope (BE Meyers, Redmond, WA). Oxygenated Ringer’s solution was introduced continuously to the superfusion chamber, and the control Ringer’s contained 108 mM NaCl, 2.5 mM KCl, 1.2 mM MgCl2, 2 mM CaCl2, and 5 mM Hepes (pH 7.7). All chemicals were dissolved in control Ringer’s solution.

A photostimulator was used to deliver light spots (of diameter 600-1,200 μm) to the retina via the epi-illuminator and the objective lens of the microscope. The intensity of unattenuated (log I = 0) 500 nm light was 2.05 × 107 photons μm−2 sec−1. Since we delivered an un-collimated stimulus light beam through an objective lens with large numerical aperture (Zeiss 40×/0.75 water), the incident light entered the retinal slice in many directions, and thus the effect of photoreceptor self-screening was minor

Dual or single voltage-clamp recordings were made with an Axopatch 700A amplifier connected to a DigiData 1200 interface and pClamp 10 software. Patch electrodes of 5 MΩ tip resistance (when filled with an internal solution containing 118 mM Cs methanesulfonate, 12 mM CsCl, 5 mM EGTA, 0.5 mM CaCl2 , 4 mM ATP, 0.3 mM GTP, 10 mM Tris, 0.8 mM Lucifer yellow or sulphorhodamine, and adjusted to pH 7.2 with CsOH) were made with Narishige or Sutter patch electrode pullers. The chloride equilibrium potential, ECl , with this internal solution was approximately −60 mV. The equilibrium potential of cation current (EC) was determined by the reversal potential of glutamate induced current in morphologically-identified bipolar cells in Ringer’s containing 2 mM Co2+. 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 by 10 mV.

Three-dimensional cell morphology was visualized in living retinal slices (250-300 μm in thickness) through the use of Lucifer yellow fluorescence with a confocal microscope (Zeiss 510). Images were acquired by using a x40 water immersion objective (n.a.= 0.75), the 458 nm excitation line of an argon laser, and a long pass 505 nm emission filter. Consecutive optical sections were superimposed to form a single image using the Zeiss LSM-PC software, and these compressed image stacks were further processed in Adobe Photoshop 6.0 to improve the contrast.

For immunocytochemistry, retinas were fixed in 4% paraformaldehye in phosphate-buffered saline (PBS; pH 7.4) for 30-60 minutes at room temperature, and then extensively rinsed with PBS. Whole-mount retinal tissue was blocked with 3% donkey serum in PBS with 0.5% Triton X-100 and 0.1% sodium azide from 2 hours to overnight to reduce nonspecific labeling. The tissue was then incubated in primary antibody in the presence of 1% donkey serum/PBS with 0.5% Triton X-100/ 0.1% sodium azide for 3-10 days at 4°C. Controls lacking primary antibodies were blank. After extensive washing with PBS containing 0.5% Triton X-100/ 0.1% sodium azide the tissue was incubated overnight with immunofluorescent secondary antibody. After further rinsing the tissue was mounted with Vectrashield. The specimens were then observed with a confocal laser-scanning microscope (Zeiss LSM510). Antibodies against GluR4 and Goα were obtained from Chemicon International (Temecula, CA) and used at a dilution of 1:1000. Secondary antibodies were donkey conjugated CY3 (Jackson ImmunoResearch, West Grove, PA) and Alexa 488 (Molecular Probes), used at a dilution of 1:100. TOPRO3 (0.01 μl/ml), a nuclear dye used to label cell bodies in ONL, was obtained from Molecular Probes (Eugene, OR).

3. Results

Voltage steps in rods drive three types of depolarizing bipolar cells (DBCs)

In order to study photoreceptor inputs to DBCs, we made dual whole-cell voltage clamp recordings from 58 rod/DBC pairs in dark-adapted salamander retinal slices. Rods and DBCs were identified by their characteristic light response waveforms and morphology (revealed by Lucifer yellow fluorescent images). In 25 rod/DBC pairs, voltage steps in rods did not elicit any postsynaptic responses, whereas in the remaining 33 pairs, three types of DBCs (DBCI (n=11), DBCII (n=10) and DBCIII (n=12)) were distinguished, based on their postsynaptic current responses to voltage clamp steps in the rods. Figure 1 shows an example of the rod/DBCI pairs filled with Lucifer yellow (a), the simultaneous current responses to a light step, with the rod voltage held at −40 mV and the DBCI at various holding potentials (b); and the DBCI current responses at various holding potentials to a depolarizing and a hyperpolarizing voltage step in the rod (c). Current-voltage (I-V) relations of the light- and voltage-evoked currents are given in Figure 1d. Transient outward currents were observed in the DBCI at the onset of the depolarizing voltage step and at the offset of the hyperpolarizing voltage step, and a transient inward current was seen at the hyperpolarizing voltage step onset. Sustained voltage steps in the rod generate transient current responses in DBCs because the voltage step in a rod results in current flow to adjacent rods via rod-rod coupling (Attwell & Wilson, 1980;Zhang & Wu, 2005) and Ih in the unclamped adjacent rods shapes the sustained current flow into transient voltage changes at the voltage step onset and offset (Attwell & Wilson, 1980;Barrow & Wu, 2009). Consequently, these rods transiently alter the glutamate release rate and result in transient postsynaptic responses in the DBCs, which receive convergent synaptic inputs from these rods (Wu et al., 2000). [Note that rod-cone coupling is much weaker than rod-rod coupling in dark-adapted salamander retina (Attwell et al., 1984) and thus rod voltage steps elicit very small voltage changes in adjacent cones whose contribution to the rod depolarization-induced DBC signals is insignificant (Pang et al., 2008)]. The I-V relations indicate that the transient outward currents are accompanied by a conductance increase (positive I-V slope) and the voltage- and light-evoked sustained inward currents are accompanied by a conductance decrease (negative I-V slope), and all current responses reversed near +20 mV. We obtained similar responses in all 11 rod/DBCI pairs and the average (±s.d.) reversal potential of the I-V relations is +16±7 mV. These results suggest that the light- and rod voltage-elicited currents in DBCIs are mediated by the same synaptic mechanism. As suggested by results in the next section, all light- and rod voltage-evoked currents in DBCIs are sensitive to L-AP4, thus they are mediated by the mGluR6 receptors (Nawy & Jahr, 1990).

Figure 1. Light- and rod voltage-induced responses in DBCI.

Figure 1

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(a): a rod/ DBCI pair filled with Lucifer yellow in a salamander retinal slice; (b) simultaneous current responses of the rod (held at −40 mV, upper trace) and the DBCI (at various holding potentials, lower traces) to a light step (500nm, −3); (c) current responses of the DBCI at various holding potentials to a positive voltage step (from −40mV to 20 mV) and a negative voltage step (from −40mV to −100 mV) in the rod; (d) current-voltage relations of the light-evoked currents (triangles), the positive step onset (filled dots), positive step offset (open dots), negative step onset (filled squares) and negative step offset (open squares) components of the rod-elicited responses of the DBCI.

Figure 2 shows the morphology and responses of a rod/DBCII pair obtained by the same protocols and similar dark-adapted conditions as for the rod/DBCI pair in Figure 1. Transient inward currents were observed in the DBCII (as opposed to the transient outward currents in DBCIs) at the onset of the depolarizing voltage step and the offset of the hyperpolarizing voltage step. The I-V relations (d) show that the transient inward currents as well as the light-evoked sustained inward current are accompanied by a conductance increase (positive I-V slope). Since depolarizing voltage step onset and hyperpolarizing voltage step offset in a rod cause transient depolarizations in the rod network (Attwell & Wilson, 1980) and light causes rod hyperpolarization, our results showing that the voltage- and light-elicited postsynaptic currents share the same sign of conductance change in the DBCII suggest that the two currents must be mediated by two different synaptic mechanisms. Similar results were obtained in all 10 dark-adapted rod/DBCII pairs, the reversal potential of the rod-voltage-elicited transient inward currents ranges between −15 and +5 mV, and that of the light-evoked currents ranges between −25 and −5 mV.

Figure 2. Light- and rod voltage-induced responses in DBCIIs.

Figure 2

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(a): a rod/ DBCII pair filled with Lucifer yellow in a salamander retinal slice; (b) Current responses of the DBCII at various holding potentials to a positive voltage step (from −40mV to 20 mV) and a negative voltage step (from −40mV to −100 mV) in the rod; (c-d) simultaneous current responses of the rod (c) and the DBCII (d) to a light step (500nm, −3), with the rod voltage held at −40 mV and the DBCII voltage at various holding potentials; (e) current-voltage relations of the light-evoked currents (triangles), the positive step onset (filled dots), positive step offset (open dots), negative step onset (filled squares) and negative step offset (open squares) components of the rod-elicited responses of the DBCII.

Figure 3 shows the morphology and responses of a rod/DBCIII pair obtained by the same protocols and adaptation conditions as for the rod/DBCI pair. Transient inward currents were observed in the DBCIII at the onset of the depolarizing voltage step and at the onset and offset of the hyperpolarizing voltage step. The I-V relations (d) show that the voltage-elicited transient inward currents as well as the light-evoked sustained inward current are accompanied by a conductance increase (positive I-V slope). Similar to the rod/DBCII pair, rod depolarization (positive voltage step onset and negative step offset) and rod hyperpolarization (negative voltage step onset and light) elicited postsynaptic currents of the same sign of conductance change. Thus two different synaptic mechanisms must be involved in mediating the DBCIII responses. Similar results were obtained in all 12 dark-adapted rod/DBCIII pairs, the reversal potential of the rod-voltage-elicited transient inward currents ranges between −35 and −15 mV, and that of the light-evoked currents ranges between −25 and 0 mV.

Figure 3. Light- and rod voltage-induced responses in DBCIIIs.

Figure 3

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(a): a rod/ DBCIII pair filled with Lucifer yellow in a salamander retinal slice; (b) Current responses of the DBCIII at various holding potentials to a positive voltage step (from −40mV to 20 mV) and a negative voltage step (from −40mV to −100 mV) in the rod; (c-d) simultaneous current responses of the rod (c) and the DBCIII (d) to a light step (500nm, −3), with the rod voltage held at −40 mV and the DBCIII voltage at various holding potentials; (e) current-voltage relations of the light-evoked currents (triangles), the positive step onset (filled dots), positive step offset (open dots), negative step onset (filled squares) and negative step offset (open squares) components of the rod-elicited responses of the DBCIII.

Rod and cone inputs to the light-evoked cation currents in the three types of DBCs

To determine the relative rod/cone contribution to the light responses of the three types of DBCs, we compared current responses of rods and cones with cation currents (measured at ECl) of the DBCIs, DBCIIs and DBCIIIs to a light step pair (duration of each step: 0.5 sec, wavelength: 500nm, attenuation: −2.5 log units, separation of the two steps: 12 seconds). Figure 4 shows an example of each of the five cell types, and it is evident that the rod response to the second light step is insignificantly small whereas the cone response to the second light step is about the same as the response to the first light step. The DBCI response to the second step is almost undetectable, suggesting that DBCI light response is mediated by rods, similar to the rod-dominated DBCs (DBCRs) described in earlier studies (Pang et al., 2004). The DBCII response to the second step is the same as the first response, suggesting that the DBCII light response is mediated by cones, resembling the previously reported cone-dominated DBCs (DBCCs) (Wu et al., 2000). The DBCIII response to the second step is about half that of the first light step, suggesting that DBCIII light response is mediated by mixed rod/cone inputs, similar to the previously described mixed DBCs (DBCMs) (Pang et al., 2004). Such patterns of cation current responses to the light step pair are consistent in all 11 DBCIs, 10 DBCIIs and 12 DBCIIIs. As mentioned above, 25 of the 58 rod/DBC pairs recorded in this study did not exhibit postsynaptic responses to rod voltage steps. Among these 25 DBCs, none showed rod-dominated light responses, 12 displayed cone-dominated light responses and 13 exhibited mixed rod/cone responses. Therefore according to this data pool, DBCIs account for 100% (11/(11+0)) of DBCRs, DBCIIs account for about 45% (10/(10+12)) of DBCCs, and DBCIIIs account for 48% (12/(12+13)) of DBCMs.

Figure 4. Rod/cone inputs to DBC light responses under dark-adapted conditions.

Figure 4

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Current responses of a rod, a cone, a DBCI, a DBCII and a DBCIII recorded under dark-adapted conditions at holding potential −40 mV to two light steps (duration of each step: 0.5 sec, wavelength: 500nm, attenuation: −2.5 log unit attenuation, separation of the two steps: 12 seconds). The rod response to the second light step is insignificantly small whereas the cone response to the second light step is similar to that to the first light step. The DBCI response to the second step is almost undetectable, suggesting that DBCI light response is primarily mediated by rods. The DBCII response to the second step is the same as the first response, suggesting that the DBCII light response is primarily mediated by cones. The DBCIII response to the second step is about half that of the first light step, suggesting that DBCIII light response is mediated by mixed rod/cone inputs.

mGluR6- and AMPA-receptors in three types of DBCs

We next characterized glutamate receptors in the three types of DBCs by using the mGluR6-specific agonist L-AP4 (Slaughter & Miller, 1981), an AMPA/kainate receptor-specific antagonist DNQX (Wu & Maple, 1998) and an AMPA receptor preferring desensitization blocker cyclothiazide (CTZ) (Partin et al., 1994;Yang et al., 1998;Pang et al., 2008). Figure 5 shows that 20 μM L-AP4 blocks the light-evoked cation current (a and b left), glutamate-elicited outward cation current (b right) and rod voltage-induced transient cation currents (c) in a DBCI. Similar results were obtained from all 8 DBCIs tested with L-AP4. This is consistent with the results in Figure 1 which suggest that both light- and rod voltage-induced postsynaptic currents are mediated by a single synaptic mechanism, and results in Figures 4 and 5 indicate that the mechanism is the mGluR6-mediated, sign-inverting glutamatergic transmission from rods to DBCIs. Since our experiments show no indication that DBCIs contain AMPA/kainate receptors, we did not include DNQX and CTZ in the DBCI studies.

Figure 5. Effects of pharmacological agents on DBCIs.

Figure 5

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(a) Effect of 20 μM L-AP4 on light-evoked cation current in a DBCI. (b) Light-evoked current response and current response to a 1-second puff glutamate application to the dendrites of a DBCI in the absence (upper trace) and presence of 20 μM L-AP4 + 1 mM Co2+. (c) Rod voltage-induced current responses in the absence (upper trace) and presence of 20 μM L-AP4 in a DBCI. The DBCIs were held at −60 mV, the light step (0.5 sec, 500nm, −2.5) and the positive and negative voltage steps in the rod are the same as the corresponding stimuli in Figures 1 and 4.

We then used the same protocol to study DBCII and DBCIII. Figure 6 shows that in a DBCII, 20 μM L-AP4 blocks the light-evoked inward current (a) and converted the glutamate-elicited biphasic current into an inward current (b). L-AP4 also slightly enhanced the transient inward current at the rod depolarization step onset and hyperpolarization offset (c). The residual glutamate- and rod voltage-induced responses were completely blocked by 100 μM DNQX (c). Additionally, we found that 100 μM CTZ enhanced and prolonged the transient inward current at the rod depolarization step onset and hyperpolarization offset (d). Similar results were obtained in all 7 DBCIIs tested with these drugs. Results in Figure 6 in conjunction with those in Figures 2 and 4 suggest that two different synaptic mechanisms are involved in rod/cone inputs to DBCIIs: (1) a mGluR6-mediated, sign-inverting glutamatergic synapse for the cone-mediated light response; and (2) an AMPA receptor-mediated, sign-preserving glutamatergic synapse for the rod depolarization-induced responses (at the rod depolarizing voltage onset and rod hyperpolarizing voltage offset). According to this scheme, the cone-DBCII synapse only contains mGluR6 and it mediates the DBCII’s light response, and the rod-DBCII synapse contains the AMPA receptors (not kainate receptor because of the CTZ sensitivity (Partin et al., 1993)). These AMPA receptors are only responsive to rod depolarization, because the AMPA receptors occupied by glutamate released in darkness are at the desensitized state, thus rod hyperpolarzation-induced glutamate decrease has very few open channels to close, but rod depolarization-induced glutamate increase opens more unoccupied channels.

Figure 6. Effects of pharmacological agents on DBCIIs.

Figure 6

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(a) Effect of 20 μM L-AP4 on light-evoked cation current in a DBCII. (b) Light-evoked current response and current response to a 1-second puff glutamate application to the dendrites of a DBCII in control Ringer’s (upper trace), in the presence of 20 μM L-AP4 + 1 mM Co2+ (middle trace) and in the presence of 100 μM DNQX+ 20 μM L-AP4 + 1 mM Co2+. (c) Rod voltage-induced current responses of a DBCII in control Ringer’s (upper trace), in 20 μM L-AP4 (middle trace) and in 100 μM DNQX+ 20 μM L-AP4 (lower trace). (d) Rod voltage-induced current responses of a DBCII in control Ringer’s (upper trace) and in 100 μM CTZ (lower trace). The DBCIIs were held at −60 mV, the light step (0.5 sec, 500nm, −2.5) and the positive and negative voltage steps in the rod are the same as the corresponding stimuli in Figures 1 and 4.

In the DBCIII, 20 μM L-AP4 blocks the light-evoked cation current (Figure 7a) and the glutamate-elicited initial outward cation current, without affecting the glutamate-induced late inward current (b). L-AP4 also suppressed the transient inward current at the rod hyperpolarization onset, but slightly enhanced the transient inward current at the rod depolarization onset and hyperpolarization offset (c). The residual glutamate- and rod voltage-induced responses were completely blocked by 100 μM DNQX (c). 100 μM CTZ enhanced and prolonged the transient inward current at the rod depolarization step onset and hyperpolarization offset (d). Similar results were obtained in all 6 DBCIIIs tested with these drugs. In conjunction with results in Figures 3 and 4, Figure 7 suggests that two different synaptic mechanisms are involved in rod/cone inputs to DBCIIIs: (1) a mGluR6-mediated, sign-inverting glutamatergic synapse for the mixed rod/cone-mediated light response and the rod hyperpolarization-mediated response at the rod hyperpolarizing voltage step onset; and (2) an AMPA receptor-mediated, sign-preserving glutamatergic synapse for the rod depolarization-induced responses at the rod depolarization onset and hyperpolarization offset. According to this scheme, the cone-DBCIII synapse only contains mGluR6 and it mediates the DBCIII’s light response. The rod-DBCIII synapse contains both mGluR6 and AMPA receptors (not kainate receptor because of the CTZ sensitivity (Partin et al., 1993)). The mGluR6 receptors are responsive to both rod depolarization and rod hyperpolarization (thus contribute to the DBCIII light response), and the AMPA receptors are only responsive to rod depolarization (because the AMPA receptor desensitization, as described above). This explains why L-AP4 blocks the rod hyperpolarization-induced currents but enhances the rod depolarization-induced currents in this type of DBCs.

Figure 7. Effects of pharmacological agents on DBCIIIs.

Figure 7

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(a) Effect of 20 μM L-AP4 on light-evoked cation current in a DBCIII. (b) Light-evoked current response and current response to a 1-second puff glutamate application to the dendrites of a DBCIII in control Ringer’s (upper trace), in the presence of 20 μM L-AP4 + 1 mM Co2+ (middle trace) and in the presence of 100 μM DNQX+ 20 μM L-AP4 + 1 mM Co2+. (c) Rod voltage-induced current responses of a DBCIII in control Ringer’s (upper trace), in 20 μM L-AP4 (middle trace) and in 100 μM DNQX+ 20 μM L-AP4 (lower trace). (d) Rod voltage-induced current responses of a DBCIII in control Ringer’s (upper trace) and in 100 μM CTZ (lower trace). The DBCIIIs were held at −60 mV, the light step (0.5 sec, 500nm, −2.5) and the positive and negative voltage steps in the rod are the same as the corresponding stimuli in Figures 1 and 4.

Rod/cone inputs to DBCI-III light responses under light-adapted conditions

In order to determine how rod/cone inputs and mGluR6/AMPA receptors mediate DBC responses under light-adapted conditions, we examined the light-evoked cation currents in DBCIs, DBCIIs and DBCIIIs in the presence of background light. Current responses of a rod, a cone, a DBCI, a DBCII and a DBCIII to a bright light step (2.5-sec, 500nm, −1.5 log unit attenuation) recorded before, 8 minute after and 15 minutes after the onset of an adapting background light (500nm and −2.5 log unit attenuation) are shown in Figure 8ABC. In the presence of the background light, rods maintained a steady outward current, and they exhibit a much smaller response (than that in darkness) to the light step and a cone-like waveform (with abrupt offset responses, arrows). This is because the background used adapts the rod photocurrent and enhances rod-cone coupling (Yang & Wu, 1989). The cone step responses did not change with the background light, as it is not bright enough to adapt cones (Wu & Yang, 1992). DBCI step responses in background light (mediated by cone signals via rod-cone coupling) are small, consistent with the results in Figure 4 that the DBCI light response is mediated primarily by rods. DBCII ON responses in background light are similar to the dark-adapted response, consistent with the result that DBCII light responses are mediated primarily by cones. DBCIII ON responses in background are slightly smaller than the dark-adapted responses, consistent with the results that DBCIII responses are mediated by mixed rod/cone inputs. Light-adapted DBCII and DBCIII also exhibit a transient inward current response at the light step offset, possibly mediated by the abrupt depolarizing voltage change (abrupt inward current, arrows in Figure 8) in rods via AMPA receptors (Figure 2b and 6c). Figure 8D shows that these transient OFF responses in the light-adapted DBCII and DBCIII are blocked by 100 μM DNQX, supporting the idea that they are mediated by the GluR4 AMPA receptors found at the basal junctions between rods and DBCIIs/DBCIIIs. We obtained similar results were from all 6 rods, 5 cones, 6 DBCIs, 7 DBCIIs and 5 DBCIIs in which the experiments described in Figure 8 were carried out.

Figure 8. Rod/cone inputs to DBC light responses under light-adapted conditions.

Figure 8

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Current responses of a rod, a cone, a DBCI, a DBCII and a DBCIII to a light step (2.5 sec, 500nm, −1.5) recorded before (a), 8 minutes after (b), 15 minutes after (c) and 18 minutes after (d) the onset of a adapting background light (500nm, −2.5). 100 μM DNQX was present in the bath in d. Rod responses in the presence of the adapting background light are much smaller with a cone-like waveform (with abrupt off responses, arrows), as rod photocurrent is suppressed and rod-cone coupling is enhanced by the background light. A DNQX-sensitive, transient inward current is found at the light step offset in the light adapted DBCII and DBCIII (arrowheads), but not in the light-adapted DBCI.

GluR4 receptors are located near flat synaptic contacts between rods and DBCIIs/DBCIIIs

We next used immunocytochemical methods to examine whether AMPA receptors are localized in DBC dendrites. Figure 9A presents the same Lucifer-yellow filled rod/DBC pairs as shown in Figures 1a, 2a and 3a (green) immunostained with antibodies against the GluR4 subunit (red). It is evident that GluR4-positive plaques (red) are co-localized with DBCII and DBCIII dendrites (green, to form yellow spots with the red), but not on DBCI dendrites. In triple-labeling experiments (Figure 9B, DBC dendrites labeled with Goα: red, GluR4: green, and rods labeled with neurobiotin, NB: blue), GluR4 plaques (arrows) are not located in the rod invaginations (arrowheads), but at the base of rod axon terminals, where flat synapses are normally found (Lasansky, 1978;Lasansky, 1973). We immunostained 5 DBCIs, 4 DBCIIs and 4 DBCIIs with GluR4 antibodies, and triple-labeled 6 rods with NB in conjunction with anti-Goα and anti-GluR4, and obtained similar patterns of GluR4 distribution in dendrites of the three types of DBCs and GluR4 plaque locations in rod axon terminals.

Figure 9. Immunostaining of GluR-4 in DBCs.

Figure 9

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A: Lucifer-yellow filled rod/DBC pairs in retinal slices (same as shown in Figures 1a-3a) immunostained with antibodies against GluR-4 subunits. GluR-4-positive plaques (red) are found in DBCII and DBCIII dendrites (green), but not on DBCI dendrites. B: Retinal sections triple-labeled with neurobiotin (NB, injected into rods, blue), anti-Goα (labels DBC dendrites, red) and anti-GluR4 (green). Note that the GluR4 plaques (green) do not label the rod outer/inner segments or cell body (top), do not label not the rod invaginations (arrow heads), but label DBC dendrites where basal junctions are found (arrows). Scale bars; 20 μm.

4. Discussion

Glutamatergic synaptic inputs from rods and cones to DBCs

In the tiger salamander retina, we have shown that three types of DBCs (DBCIs, DBCIIs and DBCIIIs) can be driven by voltage steps in rods. Depolarizing voltage changes in the rod (onset of a positive voltage step or offset of a negative voltage step) elicit transient outward currents, and hyperpolarizing voltage changes (offset of a positive voltage step or onset of a negative voltage step) evoke inward currents in DBCIs. These rod voltage-elicited, sign-inverting postsynaptic currents are suppressed in 20 μM L-AP4, suggesting that they are mediated by mGluR6 receptors which result in cation channel closure upon glutamate binding (Nawy & Jahr, 1991). In DBCIIs, rod depolarizations give rise to transient inward, sign-preserving postsynaptic currents, and rod hyperpolarizations generate very little current. The rod depolarization-elicited inward currents persist in 20 μM L-AP4, but are suppressed by 100 μM DNQX and enhanced by 100 μM CTZ, indicating that they are likely to be mediated by AMPA receptors (Partin et al., 1994). In DBCIIIs, both rod depolarization and rod hyperpolarization give rise to transient inward currents. The former sign-preserving current is resistant to L-AP4, suppressed by CNQX and enhanced by CTZ, and thus it is mediated by AMPA receptors. The latter sign-inverting current is suppressed by L-AP4, and hence it is mediated by mGluR6 receptors.

Previous studies have shown that there are three types of DBCs in dark-adapted tiger salamander retina: rod-, cone-dominated and rod/cone mixed DBCs (DBCR, DBCC and DBCM) (Pang et al., 2004). By analyzing responses to light steps and rod voltage changes (Figures1-4), our results indicate that DBCIs are DBCRs, DBCIIs account for about 45% of DBCCs and DBCIIIs account for 48% of DBCMs. We also found that light responses of all three types of DBCs (DBCI-III) under dark-adapted conditions can be blocked by L-AP4, consistent with the finding that light-evoked inputs to all DBCs in the salamander retina are mediated by mGluR6 receptors (Gao et al., 2000). Our results of rod voltage-, glutamate- and light-evoked responses suggest that photoreceptor inputs to DBCIs are primarily mediated by rods via mGluR6 receptors, because postsynaptic currents evoked by these three methods reverse at the same potential near EC, and they are all suppressible by L-AP4. The cone-dominated photoreceptor inputs to DBCIIs are mediated by mGluR6 receptors, as the cells’ light response as well as the outward component of the glutamate-induced current can be suppressed by L-AP4. DBCIIs also receive AMPA receptor-mediated inputs from rods, as rod depolarization- and glutamate-induced inward currents can be blocked by DNQX and enhanced by CTZ. DBCIIIs exhibit combined photoreceptor inputs of DBCIs and DBCIIs: they receive inputs from both rods and cones via mGluR6 receptors, as the light response and the outward component of the glutamate-induced current are L-AP4 sensitive. DBCIIIs also receive AMPA receptor-mediated inputs from rods, as rod depolarization- and glutamate-induced inward currents can be blocked by DNQX and enhanced by CTZ. A schematic diagram of the mGluR6 and AMPA receptor distribution in DBCI-III dendrites in rod and cone output synapses is given in Figure 10.

Figure 10. Schematic diagram illustrating synaptic inputs from rods and cones to the DBCI (left), DBCII (middle) and DBCIII (right).

Figure 10

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Black arrows: invaginating ribbon synapses, pink arrows: basal junctions. Green/yellow hexagons represent mGluR6 receptors, red hexagons represent GluR4 AMPA receptors, blue circles are synaptic vesicles and small blue dots are glutamate molecules. Thick double lines with triple hexagons represent DBC dendrites with major synaptic inputs, thin double lines with double hexagons represent DBC dendrites with medium synaptic inputs and thin single lines with single hexagons represent dendrites with minor synaptic inputs.

GluR4 AMPA receptors are located in DBCII and DBCIII dendrites at basal junctions postsynaptic to rods

Our immunocytochemical results have shown that GluR4 AMPA receptors are not located in dendrites of DBCIs, but in DBCII and DBCIII dendrites, consistent with the physiological data that only the latter two types of DBCs exhibit DNQX-sensitive, rod depolarization-induced, sign-preserving postsynaptic responses. Our triple-label results indicate that GluR4 AMPA receptors are located at DBC dendritic regions where basal junctions postsynaptic to rods are found (Lasansky, 1973). It has been shown by horse radish peroxidase staining and electron microscopic techniques that DBCs in the tiger salamander retina make synaptic contacts with rod and cones at basal junctions (Lasansky, 1978). Our findings in this report suggest that a substantial portion of the basal junctions between rods and DBCIIs/DBCIIIs use GluR4 AMPA receptors to transmit synaptic signals (see Figure 10).

In an earlier study, we reported that the GluR4 AMPA receptors between rods and rod-dominated hyperpolarizing bipolar cells (HBCRs) at the invaginating ribbon synapses evade desensitization in darkness by low frequency, multiquantal release so that release events do not desensitize one another (Pang et al., 2008). In this report, our evidence suggests that GluR4 AMPA receptors in basal junctions between rods and DBCIIs/DBCIIIs are desensitized in darkness. This indicates that mechanisms of glutamate release at basal junctions and invaginating ribbon synapses are very different. Although synaptic vesicles have been observed near basal junctions in photoreceptor synaptic terminals, they are normally not at the “primed” locations next to the presynaptic membrane (Lasansky, 1978). It is possible that glutamate release from these basal junctions is not mediated by low frequency discrete vesicular events, but instead by a continuous release mechanism that results in GluR4 AMPA receptor desensitization in darkness. Consequently, light-evoked or voltage-elicited rod hyperpolarization under dark-adapted conditions does not generate postsynaptic voltage changes in these GluR4 receptor-containing basal junctions.

Because of lack of specific antibodies, we are unable to localize mGluR6 receptors in the salamander retina. However, physiological results indicate that these receptors must exist in dendrites of all DBCs. They should be located primarily at DBCI dendrites postsynaptic to rods, DBCII dendrites postsynaptic to cones, and DBCIII dendrites postsynaptic to both rods and cones (Figure 10). Since mGluR6 receptors mediate closure of cation channels upon glutamate binding and exhibit little desensitization (Nawy & Jahr, 1991;Shiells & Falk, 2002), sign-inverting postsynaptic responses can be generated by light or rod voltage changes (either depolarization or hyperpolarization) at either basal junctions or invaginating ribbon synapses under dark- or light-adapted conditions. Further studies are needed to verify the proposed scheme of mGluR6 distribution in the salamander retina.

Alternative synaptic pathways for the DNQX-sensitive signals in DBCs

Although our results suggest that the rod-depolarization-induced inward currents in DBCIIs and DBCIIIs are likely to be mediated by AMPA receptors in DBCII and DBCIII dendrites postsynaptic to rods, it is important to consider alternative interpretations. For example, rod depolarization also induces transient inward currents (depolarizations) in horizontal cells (HCs) which are DNQX-sensitive (Yang et al., 1998;Attwell et al., 1983), and thus the rod-depolarization-induced inward currents in DBCIIs and DBCIIIs could be mediated indirectly by AMPA receptors in HCs via sign-preserving synapses. It has been proposed that HCs mediate sign-preserving inputs to DBCs via three possible synaptic pathways: (1) the HC→cone→DBC feedback synaptic pathway (Baylor et al., 1971;Wu, 1992), (2) the HC→DBC chemical synapse (Dowling & Werblin, 1969;Yang & Wu, 1991) and (3) the HC→DBC electrical synapse (Zhang & Wu, 2009). Our results in Figures 6 and 7 suggest that pathway (1) is unlikely to be involved, because both rod-depolarization- and glutamate-puff-induced inward currents in DBCIIs and DBCIIIs persist in L-AP4, which blocks the cone→DBC segment of the HC→cone→DBC feedback pathway. Additionally, Figure 6b and 7b indicate that pathway (2) may not be a viable explanation either, because the glutamate-induced inward currents in DBCIIs and DBCIIIs are present in Co2+ which blocks the HC→DBC chemical synaptic transmission. Therefore the DNQX-sensitive AMPA receptors that mediate glutamate-induced inward currents in DBCs are located in DBCs, rather then in HCs connected by chemical synapses. Finally, it is hard to completely rule out the HC contribution to the DNQX-sensitive, rod-depolarization-induced signals in DBCs via the HC→DBC electrical synapse, since electrical synapses can be Co2+-insensitive (Lu & McMahon, 1997), and dye coupling have been found between HCs and DBCCs in the salamander retina (Zhang et al., 2006;Zhang & Wu, 2009). It is possible that the rod-depolarization-induced HC transient signals “leak” into the DBCs via gap junctions. However, such HC contribution should be minor because Figures 2 and 3 show that the rod-depolarization-induced currents in DBCIIs and DBCIIIs can be reversed by DBC polarizations. It is unlikely that changes in DBC holding potentials can polarize the HCs (via electrical synapses) enough to reach the reversal potential of the rod-depolarization-induced current in the latter cells, and thus the majority of the current must be originated from the recorded DBCs.

In addition to HCs, amacrine cells (ACs) may be involved in mediating the rod-depolarization-induced signal in DBCs, as they make feedback chemical synapses on DBC axon terminals (Wong-Riley, 1974), and mediate ON and OFF channel crossover inhibition (Molnar et al., 2009;Werblin, 2010). Based on the same argument as for the HC→DBC chemical synapse discussed above, the glutamate-induced AMPA current in the presence of Co2+ we observed in DBCII-IIIs are unlikely to be mediated by ACs via chemical synapses. Moreover, we could never induce significant postsynaptic signals in any ACs by injecting current steps in the rod (unpublished data), and thus AC contributions to the rod-depolarization-induced DBC signals must be negligible.

AMPA receptors in DBCs are silent under dark-adapted conditions and mediate transient off responses in light-adapted retinas

Glutamate is released continuously from rods and cones in darkness (Copenhagen & Jahr, 1989), and the AMPA receptors activated by such tonic glutamate flow in DBCII and DBCIII dendrites at the rod basal junctions are at the desensitized state. Therefore under dark-adapted conditions, reduction of glutamate release induced by rod hyperpolarization (with light or negative voltage changes) does not elicit AMPA receptor-mediated postsynaptic currents. This explains why light responses of all DBCs in dark-adapted salamander retina are sensitive to L-AP4, but not to DNQX. However, when rods are abruptly depolarized by positive voltage step onsets or negative voltage step offsets, AMPA receptor-mediated postsynaptic responses (transient inward currents that can be blocked by DNQX and enhanced by CTZ) are observed in DBCIIs and DBCIIIs. This is because an abrupt rod depolarization causes an abrupt, transient increase of glutamate release (by activating calcium channels in rods (Bader et al., 1982), increased loading of presynaptic vesicles at the tonic hyperpolarized state (Jackman et al., 2009), and/or regenerative off-overshoot responses in rods (Wu, 1988)), that binds to the unoccupied AMPA receptors, resulting in a transient increase of inward current. An interesting observation is that such transient inward currents are completely blocked by DNQX, but only moderately enhanced by CTZ (Figures 6cd and 7cd). This suggests that the AMPA receptors activated by the transient increase of glutamate release are only partially desensitized, as the transient increase of glutamate release does not provide enough time for full mutual desensitization among various discrete vesicular events (Grosskreutz et al., 2003;Pang et al., 2008). It is important to note that abrupt depolarization does not occur naturally in dark-adapted rods, because light only hyperpolarizes rods and the depolarizing rod voltage recovery after light is turned off is very slow (Wu, 1987a). Therefore AMPA receptors in dark-adapted DBCs are normally silent, and their actions are observable only when rods are abruptly depolarized by experimental voltage clamp steps. Under light-adapted conditions, however, rod-cone coupling is much stronger (Yang & Wu, 1989), and thus the abrupt depolarization in cones at a light step offset results in an abrupt depolarization (an abrupt inward current under voltage clamp conditions, Figure 8 arrows) in rods. This abrupt rod depolarization under physiological conditions is likely to be more pronounced than the abrupt inward current under voltage clamp, because a regenerative off-overshoot depolarizing response is present in a subpopulation of rods (Wu, 1988). The abrupt rod depolarization activates the AMPA receptors at the rod basal junctions and triggers a transient depolarization (a transient inward current under voltage clamp, Figure 8 arrowheads) at light step offsets in DBCIIs and DBCIIIs.

In figure 8, we showed that abrupt rod inward currents (depolarization under physiological conditions) at light offsets are observed in the presence of background light, but not in darkness. We attribute this to the increased rod-cone coupling in light, a phenomenon reported in the salamander retina over twenty years ago (Yang & Wu, 1989). More recently, a report suggests that rod-cone coupling in fish and mouse retinas is stronger at night and in darkness (Ribelayga et al., 2008), a phenomenon that has not been replicated by all researchers (Postma and Paul, personal communication, ARVO abstract 2046, 2010). The difference between the two studies may be due to species difference or difference in lighting conditions. Nevertheless, our results in Figure 8 clearly demonstrate that rod responses in background light better resemble the cone responses (with obvious off responses), consistent with the report that rod-cone coupling is enhanced by light in the tiger salamander retina.

We showed that the transient inward current at light step offsets in light-adapted DBCIIs and DBCIIIs can be blocked by DNQX, consistent with the suggestion that they are mediated by AMPA receptors in the rod basal junctions. It has been shown that the hyperpolarizing light responses of HBCs can be blocked by DNQX, and that the HBC OFF response contributes to the transient OFF responses in third-order cells (Wu & Maple, 1998). In amacrine cells and ganglion cells, the transient ON responses are L-AP4-sensitive and the transient OFF responses are DNQX sensitive (Hensley et al., 1993), and thus the traditional view is that the ON responses of these third-order neurons are mediated by DBCs and the OFF responses are mediated exclusively by HBCs. Our present study suggests that this view needs revision as AMPA receptors in subpopulations of DBCs may significantly contribute to the DNQX-sensitive OFF transient responses in light-adapted third- and higher-order visual neurons.

Acknowledgements

We thank Roy Jacoby, Cameron Cowan and David Simon for critically reading this manuscript. This work was supported by grants from NIH (EY004446, EY019908), NIH Vision Core (EY 02520), the Retina Research Foundation (Houston), and the Research to Prevent Blindness, Inc.

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