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Neuroscience |
1 Department of Ophthalmology and Visual Sciences, Washington University, St Louis, MO 63110, USA
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(Received 14 December 2005;
accepted after revision 23 January 2006;
first published online 26 January 2006)
Corresponding author P. D. Lukasiewicz: Department of Ophthalmology, Campus Box 8096, Washington University School of Medicine, 660 S. Euclid Avenue, St Louis, MO 63110, USA. Email: lukasiewicz{at}vision.wustl.edu
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Introduction |
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Determining the relative contributions of GABAA, GABAC and glycine receptors to L-IPSCs is important since these functionally diverse receptors (Amin & Weiss, 1994; Feigenspan & Bormann, 1994; Lukasiewicz & Shields, 1998; Cui et al. 2003; Frech & Backus, 2004) could each distinctly affect light-evoked inhibition. GABAC receptors are more sensitive to GABA and mediate more slowly decaying responses than GABAA receptors (Amin & Weiss, 1994; Feigenspan & Bormann, 1994; Lukasiewicz & Shields, 1998) and retinal glycine receptors, which also mediate rapidly decaying responses, have been shown to have slower responses than GABAA receptors (Frech & Backus, 2004).
Previous studies suggest that electrically and glutamate-evoked inhibition to rod bipolar cells is mediated by GABAA (Singer & Diamond, 2003) and/or GABAC receptors (Hartveit, 1999), but there is disagreement as to their relative roles. While the synaptic contacts to rod bipolar cells from GABAergic A17 amacrine cells have been well characterized (Nelson & Kolb, 1985; Sandell et al. 1989), less is known about synaptic inputs from other types of GABAergic and glycinergic amacrine cells.
Using receptor-specific blockers and mice that lack GABAC receptors (McCall et al. 2002), we determined how these functionally distinct receptors contribute to L-IPSCs in rod bipolar cells and how inhibition mediated by these different receptors affected the output of rod bipolar cells. We demonstrate, for the first time, the relative contributions of GABAC, GABAA and glycine receptors to light-evoked inhibition to rod bipolar cells. GABAC receptors were most effective in reducing rod bipolar cell output, while GABAA and glycine receptors made smaller contributions. We also found that GABAergic inhibitory inputs are modulated by connections between GABAergic amacrine cells, mediated by GABAA receptors. Our findings show that rod bipolar cells receive three distinct types of inhibition, which differentially modulate rod bipolar cell output.
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Methods |
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Animal protocols were approved by the Washington University School of Medicine Animal Studies Committee. Two types of mice were used. Wild-type (WT) mice, C57BL/6J strain (Jackson Laboratories; Bar Harbour, ME, USA), and GABAC
1 null mice (crossed onto the C57BL/6J background) that lacked functional GABAC receptors (GABACR null mice) (McCall et al. 2002). The experimental techniques were similar to those described for studies with ferret and mouse retinal slices (Shields et al. 2000; McCall et al. 2002). Briefly, mice of 28–90 days of age were killed by exposure to carbon dioxide gas in a rising concentration, and their eyes enucleated. The cornea, lens and the vitreous were removed. The eyecup was incubated for 20 min in dissection and storage solution (see ‘Solutions and drugs’) with 0.5 mg ml–1 hyaluronidase (Sigma, St Louis, MO, USA) to remove vitreous adhering to the retina. After incubation, the hyaluronidase solution was replaced with cold, oxygenated storage solution and the retina dissected out of the eyecup. Slices (250 µm thick) were prepared from the isolated retina, as previously described, and maintained in oxygenated storage solution at room temperature (Werblin, 1978; McCall et al. 2002). Mice were dark-adapted overnight, and all dissection and recording procedures were performed under infrared illumination to preserve the light sensitivity of the preparations.
Whole-cell recordings
Whole-cell patch recordings were made from bipolar cells and amacrine cells from mouse retinal slices as previously described (McCall et al. 2002). IPSCs were recorded from retinal bipolar cells voltage clamped to 0 mV, the reversal potential for currents mediated by non-selective cation channels. At this holding potential, we found that voltage-gated currents in rod bipolar cells were run down after 30–90 s, presumably attributable to the lack of ATP and GTP in our recording pipettes. Therefore, in our rod bipolar cell recordings it is unlikely that voltage-gated Ca2+ channels were activated, and glutamate release from the recorded rod bipolar cell is likely to be minimal. EPSCs from amacrine cells were recorded from neurones voltage clamped to –60 mV, the reversal potential for chloride currents. All recordings were made at 32°C. Liquid junction potentials of 15 mV were corrected at the beginning of each recording by adjusting the command voltages.
The recording procedures and microscope system have been described in detail previously (Lukasiewicz & Roeder, 1995). Electrodes were pulled from borosilicate glass (1B150F-4; World Precision Instruments, Sarasota, FL, USA) on a P97 Flaming/Brown puller (Sutter Instruments, Novato, CA, USA) and had resistances of < 5 M
. Patchit software (White Perch Software, Somerville, MA, USA) was used to generate voltage command outputs, acquire data, and gate the drug perfusion valves. The data were digitized and stored with a Pentium personal computer using a Labmaster DMA data acquisition board (Scientific Solutions, Solon, OH, USA). Responses were filtered at 1 kHz with the four-pole Bessel filter on an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA, USA) and sampled at 2 kHz. The slice and bathing solutions were heated by thin stage and inline heaters (Cell Microcontrols, Norfolk, VA, USA). The temperature of slices on the recording stage was controlled by a Cell Microcontrols temperature control system.
Morphological identification of retinal cell types
Bipolar cells and amacrine cells were identified by their characteristic morphology and cell stratification within the ‘on’ and ‘off’ sublaminae of the retinal inner plexiform layer (IPL) after labelling with Lucifer yellow (0.05%) or sulforhodamine B (0.005%), which were included in the recording electrode (Euler & Wässle, 1998; Shields et al. 2000). Our morphological identification of mouse rod bipolar cells was consistent with previous reports (Ghosh et al. 2004), where all rod bipolar cells had somas high in the inner nuclear layer (INL), and axons ending in varicosities near the ganglion cell layer (GCL) (Fig. 1C). We showed previously that the morphology of the rod bipolar cells was the same in WT and GABACR null mice (McCall et al. 2002). Although a previous report suggested that there were two distinct populations of rod bipolar cells (Pang et al. 2004), we saw no evidence for this, in agreement with other previously published reports (Ghosh et al. 2004; Pignatelli & Strettoi, 2004). We also recorded from morphologically distinct bipolar cells with axon terminals ramifying near the GCL, but these cells were clearly type 8 or 9 cone bipolar cells (Ghosh et al. 2004), and not rod bipolar cell as evidenced by their broad axonal arbors and lack of glycinergic inhibition. A17 amacrine cells (Fig. 6C) were identified as having highly branched, wide-field dendritic trees that end at the border of the IPL and GCL to receive inputs from rod bipolar cells (Nelson & Kolb, 1985; Menger & Wässle, 2000). AII amacrine cells (Fig. 6C) were identified as having lobular appendages in the ‘off’ sublamina of IPL and more fine processes in the ‘on’ sublamina (Veruki & Hartveit, 2002).
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The dissection and storage solution contained (mM): 137 NaCl, 2.5 KCl, 1 MgCl2, 2.5 CaCl2, 28 glucose and 10 HEPES, adjusted to pH 7.4 with NaOH and bubbled with oxygen. The extracellular recording solution contained (mM): 125 NaCl, 2.5 KCl, 1 MgCl2, 1.25 NaH2PO4, 2 CaCl2, 20 glucose and 26 NaHCO3 and was bubbled with carbogen (95% O2–5% CO2). The intracellular solution contained (mM): 120 caesium gluconate, 1 CaCl2, 1 MgCl2, 10 Na-Hepes, 11 EGTA, 10 TEA-Cl, and was adjusted to pH 7.2 with CsOH. The calculated chloride equilibrium potential (ECl) for recordings was –59.3 mV. To isolate receptor types, strychnine (500 nM) was used to block glycine receptors, bicuculline methobromide (50 µM, RBI, Natick, MA, USA) to block GABAA receptors and (1,2,5,6-tetrahydropyridine-4yl)methylphosphinic acid (TPMPA, 50 µM, RBI) to block GABAC receptors. Antagonists were applied to the slice under study by a gravity-driven superfusion system previously described (Lukasiewicz & Roeder, 1995). Unless otherwise indicated, all chemicals were obtained from Sigma.
Light stimulation
To evoke L-IPSCs and L-EPSCs, full-field light stimuli were generated using a light-emitting diode (LED, Agilent HLMP-3950, peak wavelength (
peak) = 565 nm, Palo Alto, CA, USA) that activated both cones and rods and was positioned near the microscope stage. The intensity of the unattenuated light was 1.85 x 105 photons µm–2 s–1, and was used at –2 log units. This intensity of light should primarily activate rod photoreceptors (Xin & Bloomfield, 1999). Light intensity was controlled by varying the current through the LED.
Data analysis
Tack (White Perch Software, Somerville, MA, USA) and Clampfit (Axon Instruments) software were used to average records and to measure the charge transfer (Q) of the light-evoked current responses for each cell. Unless otherwise stated, all analysis and traces displayed were from the average of two independent traces. Student's t tests (two-tailed, unequal variance) were used to compare these response parameters from wild-type and GABACR null neurones. Student's paired t tests were used to compare values between conditions for the same cell. To test the significance of differences among isolated GABAA, GABAC and glycine receptor-mediated currents we used ANOVA and Scheffé's post hoc test. Differences were considered significant when P
0.05. All data are reported as mean ± standard error of the mean (S.E.M.).
Spontaneous IPSCs (sIPSCs) were recorded from the dark-adapted retina, in the absence of light stimuli, and were analysed using Mini Analysis (Synaptosoft, Decatur, GA, USA). sIPSCs were selected so that the rise and decay phases did not contain any overlapping events. For each individual sIPSC the amplitude was measured. The distributions of sIPSC amplitude values were compared using the Kolmogorov-Smirnov test (K-S). To compute the average sIPSC the events were aligned by the 50% rise time.
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Results |
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To determine the components of light-evoked inhibition of rod bipolar cells, we recorded L-IPSCs from rod bipolar cells, in the absence or presence of antagonists, from WT and GABACR null mice. L-IPSCs were recorded from morphologically identified rod bipolar cells (Fig. 1C) that were voltage clamped to 0 mV, the reversal potential for the L-EPSCs. In the absence of inhibitory receptor antagonists L-IPSCs represent the summation of all light-evoked inhibitory input to the rod bipolar cell (Fig. 1A). The L-IPSCs consisted of two components; the initial 50 ms of the response was composed of discrete, fast IPSCs, which varied somewhat between trials (Fig. 1A), followed by a slowly rising and decaying response component over the next 300 ms (Fig. 1A and B). The L-IPSCs were completely eliminated in the presence of the antagonists strychnine, bicuculline and TPMPA (Fig. 1B), indicating that they were mediated exclusively by the GABAA, GABAC and glycine receptors.
To determine the contributions of glycine, GABAA and GABAC receptors to these L-IPSCs, we recorded L-IPSCs from rod bipolar cells in the presence of different combinations of antagonists to isolate the glycine, GABAA and GABAC receptor-mediated currents. Figure 2 shows that L-IPSCs mediated by all three types of isolated inhibitory receptors could be recorded from rod bipolar cells. The glycine receptor-mediated component of the L-IPSCs (Fig. 2A), recorded in the presence of bicuculline and TPMPA to block GABAA and GABAC receptors, respectively, was composed of many small discrete events. The GABAA receptor-mediated component of the L-IPSCs (Fig. 2B), recorded in the presence of strychnine to block glycine receptors and TPMPA, also consisted of many discrete events. In contrast, the GABAC receptor-mediated component of the L-IPSCs, recorded in the presence of strychnine and bicuculline (Fig. 2C), was a large, slowly rising and decaying current devoid of brief, discrete events.
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Inhibition between amacrine cells reduces rod bipolar cell L-IPSCs
If there were no inhibitory network interactions, then blocking glycine and GABAA receptors should decrease the L-IPSCs because two components of the total current were removed. Instead, as shown in Fig. 3A, we saw an increase in L-IPSC charge transfer (64.3 ± 38.8%, n= 11), suggesting that the GABAC receptor-mediated input was increased by the addition of strychnine and bicuculline. Serial inhibitory interactions between amacrine cells can regulate their GABAergic and glycinergic outputs in addition to direct amacrine cell inputs to rod bipolar cells. Blocking the glycine and GABAA receptors on amacrine cells (Wässle et al. 1998) could lead to disinhibition of amacrine cells that mediate GABA and glycine signalling to rod bipolar cells, as observed in salamander retina (Zhang et al. 1997; Roska et al. 1998).
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To determine whether GABAergic and/or glycinergic amacrine cell inputs inhibited rod bipolar cells, we applied strychnine and bicuculline separately. When glycine receptor-mediated signalling was blocked by strychnine, the L-IPSC Q was reduced (Figs 3B and 5C). However, when GABAA receptor-mediated signalling was blocked by bicuculline, the L-IPSC Q was significantly increased in rod bipolar cells (Figs 3C and 5D). Although rod bipolar cells contain GABAA receptors that would decrease inhibition when blocked, the increase in L-IPSCs with bicuculline application suggests that the net effect of blocking GABAA receptor-mediated signalling was to disinhibit amacrine cells, which increased inhibitory amacrine cell input to rod bipolar cells. Thus GABAA receptor-mediated signalling, in contrast to glycine receptor-mediated signalling, between amacrine cells limits rod bipolar cell inhibition.
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GABAA and glycine receptors are not up-regulated in GABACR null mice
Figure 4A shows an L-IPSC recorded from a GABACR null mouse. When strychnine and bicuculline were added to the bathing solution to block glycine and GABAA receptors, the L-IPSC was completely eliminated, demonstrating that these responses were mediated only by glycine and GABAA receptors. The GABACR null mouse allowed us to isolate the GABAA and glycine receptor-mediated components of the L-IPSCs from the GABAC receptor component, present in WT mice.
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We also compared GABAA and glycine receptor-mediated spontaneous IPSCs (sIPSCs) in WT and GABACR null mice, to confirm that the elimination of GABAC receptors did not result in compensation. We found that the averaged GABAA and glycine receptor-mediated sIPSCs recorded from WT and GABACR null mice were identical (Fig. 4D, inset). Comparisons of the amplitudes for the GABA and glycine receptor-mediated sIPSCs confirmed that there were no differences between WT and GABACR null mice. The distributions of amplitudes for glycine and GABAA receptor-mediated sIPSCs (Fig. 4D) show that there were no significant differences in amplitude for glycine receptor-mediated sIPSCs (WT 9.7 ± 0.5 pA, N= 5 cells, n= 83 events; null 10.5 ± 0.5 pA, N= 9 cells, n= 110 events; not significant (n.s.) K-S P > 0.6) or GABAA receptor-mediated sIPSCs (WT 6.2 ± 0.1 pA, N= 6 cells, n= 675 events; null 6.3 ± 0.2 pA, N= 3 cells, n= 297 events; n.s. K-S P > 0.1). These data support the idea that glycine or GABAA receptor expression was unchanged by the elimination of GABAC receptors. Thus the differences observed in WT and GABACR null mice can be attributed to the absence of GABAC receptors.
GABAA receptor-mediated signalling between amacrine cells had no effect on rod bipolar cell L-IPSCs in GABACR null mice
To determine whether glycinergic and/or GABAergic amacrine cells mediated the disinhibition observed in rod bipolar cells from WT mice, we applied strychnine and bicuculline separately to rod bipolar cells from GABACR null mice. When glycine receptor-mediated signalling was blocked by strychnine, the L-IPSC Q was reduced in rod bipolar cells from GABACR null mice (Fig. 5A and C), similar to the effects observed in WT mice (Fig. 5C). However, when GABAA receptor-mediated signalling was blocked by bicuculline, the L-IPSC Q in GABACR null mice was also decreased (Fig. 5B and D), in contrast to the bicuculline-dependent L-IPSC increase observed in rod bipolar cells from WT mice (Fig. 5D). This suggests that blocking GABAA receptor-mediated signalling disinhibited GABAergic amacrine cells in WT mice, which increased amacrine cell input to GABAC receptors. This increase was not observed in GABACR null mice, which lack GABAC receptors. We also saw a similar decrease in Q by bicuculline in WT mice, when TPMPA was first applied to block GABAC receptors.
A limitation of our preparation is that inhibitory blockers affect both the postsynaptic rod bipolar cell as well as the presynaptic amacrine cells. Therefore it is possible that a decrease in light-evoked inhibition by blocking postsynaptic receptors on rod bipolar cells masked an effect of presynaptic receptors on amacrine cells. We find that the net effect of blocking GABAA and glycine receptors in the GABACR null mouse decreases inhibition, and so unlike previous findings in salamander (Zhang et al. 1997; Roska et al. 1998), any presynaptic effects would be small.
Rod bipolar cell output is shaped by distinct inhibitory circuits
Our data suggest a model circuit in which rod bipolar cells receive inputs from GABAergic and glycinergic amacrine cells that modulate the release of glutamate onto AII and A17 amacrine cells. Our results also suggest that GABAergic amacrine cells receive input from other GABAergic amacrine cells, whereas the glycinergic amacrine cells do not. In contrast to the serial inhibitory networks proposed in salamander (Zhang et al. 1997; Roska et al. 2000), we saw no evidence for serial connections between glycinergic and GABAergic amacrine cells that increased inhibition at rod bipolar cell terminals. Instead, blocking glycinergic inhibition decreased light-evoked inhibition in rod bipolar cells, suggesting that glycine receptors were not involved in serial, disinhibitory circuits in the inner retina.
Our model circuit predicts that GABAC, and to a lesser extent glycine and GABAA, receptors modulate glutamate release from rod bipolar cells. In addition, GABAA receptor signals decrease GABAC receptor-mediated inhibition through serial connections between GABAergic amacrine cells. Specifically, the model predicts that blocking glycine receptors will decrease inhibition, causing increased glutamate release from rod bipolar cells. In contrast, blocking GABAA receptors will increase GABAC receptor-mediated inhibition, causing decreased glutamate release from rod bipolar cells. Furthermore, blocking GABAC receptors that are located on bipolar terminals, and not on amacrine cells, should significantly increase glutamate release from rod bipolar cells.
To test these predictions, we assessed rod bipolar cell glutamate release by recording L-EPSCs in morphologically identified AII (n= 13) and A17 (n= 7) amacrine cells (Fig. 6C). L-EPSCs were isolated by voltage-clamping amacrine cells at ECl to eliminate direct inhibitory input. L-EPSCs had both transient and sustained components, consistent with previous studies showing transient and sustained components of glutamate release from bipolar cells (von Gersdorff et al. 1998; Singer & Diamond, 2003). Figure 6A shows L-EPSCs from a WT A17 amacrine cell recorded in the absence or presence of antagonists. Blockade of glycine receptors by strychnine increased the L-EPSC Q (Fig. 6A), consistent with the removal of inhibition of glutamate release. The subsequent addition of bicuculline, to block GABAA receptors, decreased the L-EPSC Q (Fig. 6A), consistent with an increased inhibition of glutamate release from rod bipolar cells. When all inhibition was blocked with the addition of TPMPA, to block GABAC receptors, the L-EPSC Q (Fig. 6A) was dramatically increased, suggesting that GABAC receptors, in the absence of serial inhibitory circuits, played the most prominent role in rod bipolar cell inhibition. As no significant differences were observed between AII and A17 L-EPSCs (P > 0.2), the pooled results from AII and A17 amacrine cells are summarized in Fig. 6D. All of these results are consistent with our model circuit.
We determined the roles of glycine and GABAA receptors in rod bipolar cell release, in the absence of GABAC receptors, by comparing L-EPSCs from A17 and AII amacrine cells in WT and GABACR null mice. Strychnine increased the L-EPSCs in A17 amacrine cells from GABACR null mice, similar to the effect observed in amacrine cells from WT mice (Fig. 6B and D). These data suggest that glycinergic inhibition of rod bipolar cell glutamate release was not affected by the absence of GABAC receptors. By contrast, the subsequent addition of bicuculline increased the L-EPSCs from amacrine cells in GABACR null mice, instead of decreasing the L-EPSCs, as observed in amacrine cells from WT mice (Fig. 6B and D). These findings demonstrate that GABAA receptors directly modulate glutamate release from rod bipolar cell terminals, albeit to a smaller extent than GABAC receptors. The subsequent addition of TPMPA to bicuculline and strychnine did not significantly increase the L-EPSCs (Fig. 6B and D), confirming that GABAC receptors were absent.
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Discussion |
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Modulating rod bipolar cell transmitter release
GABAergic inhibition to rod bipolar cell terminals (Hartveit, 1999; Singer & Diamond, 2003) contributes to the receptive field organization of AII amacrine cells (Volgyi et al. 2002) and the light intensity–response relationship of rod bipolar cells (Euler & Masland, 2000). Our findings differ from a previous report that suggested that blocking GABAA and GABAC receptors mediated equivalent effects on AII amacrine cell surround responses (Volgyi et al. 2002). The difference may be attributed to the inability of the earlier voltage response study to distinguish presynaptic rod bipolar cell inhibition from direct inhibition to amacrine cells. Here, we show that GABAA receptors mediate serial inhibitory signalling, which obscures the direct inhibitory effects of rod bipolar cell GABAA receptors. Furthermore, we were able to determine the influence of GABAA receptors in the absence of GABAC receptors, using the GABACR null mice. We show that both the inhibition of rod bipolar cells and the modulation of their output to amacrine cells were mediated by GABAC receptors and to a smaller extent by glycine and GABAA receptors. Activation of GABAC receptors decreased rod bipolar cell outputs, in agreement with previous studies of bipolar cell to ganglion cell transmission in salamander (Dong & Werblin, 1998; Shen & Slaughter, 2001).
While glycine and GABA receptors on the dendrites of rod bipolar cells could also contribute to the L-IPSCs, most evidence argues against dendritic receptors mediating the major component of light-evoked inhibition. Studies have shown that glycine- and GABAA receptor-mediated currents in bipolar cells, evoked by agonist applications, are significantly smaller in the outer plexiform layer (OPL) than the inner plexiform layer (IPL) (Suzuki et al. 1990; Gillette & Dacheux, 1995; Maple & Wu, 1996, 1998; Bloomfield & Dacheux, 2001; Cui et al. 2003). Furthermore, GABAC receptor-mediated currents attributable to dendritic receptors were virtually absent (Euler & Masland, 2000; Shields et al. 2000). Finally, L-IPSCs were eliminated in rat rod bipolar cells that were axotomized (Euler & Masland, 2000), suggesting that most light-evoked inhibition occurs at the axon terminals.
Serial inhibitory circuits modulate rod bipolar cell transmitter release
In addition to observing modulation of rod bipolar cell glutamate release by direct inhibition, we also observed indirect modulation by serial inhibitory networks in the IPL. Serial connections between amacrine cells have been previously described as altering inputs to amacrine cells (Zhang et al. 1997) and ganglion cells in the cone-dominated circuitry of salamander (Roska et al. 1998) and feedback inhibitory inputs to bipolar cells in goldfish (Vigh et al. 2005). Here we have described, for the first time, serial inhibitory connections in the rod signalling pathway, a uniquely mammalian retinal circuit. Additionally, we have specifically defined the contribution of different inhibitory receptor types to these connections.
Although we found evidence for connections between GABAergic amacrine cells, we saw no evidence for the connections between glycinergic and GABAergic amacrine cells that had been observed in salamander. Disinhibition resulting from blocking GABAA receptor-mediated, but not glycine receptor-mediated, transmission increased GABAC receptor-mediated transmission to rod bipolar cells. It is possible that a strychnine-mediated increase in GABA receptor-mediated inputs was masked by the effect of blocking glycine receptors on rod bipolar cell terminals. We cannot rule out this possibility because of the limitations of the direct and network effects of antagonists in the retinal slice. However, our observations were different from those observed in salamander, where the net effect of strychnine was disinhibition (Zhang et al. 1997; Roska et al. 1998). A possible explanation for the different patterns of serial inhibition in mouse and salamander may be attributed to the relative lack of wide-field glycinergic amacrine cells in mouse (Menger et al. 1998) compared to salamander retina (Yang et al. 1991).
Differential roles of GABAA, GABAC and glycine receptors in the mammalian retina
Why do rod bipolar cells receive input through distinct GABAA and GABAC receptors? While anatomical evidence suggests that GABAC and GABAA receptors are located at distinct synapses in the retina (Koulen et al. 1998), it is unclear if one amacrine cell releases GABA onto both types of synapses or if distinct cells provide inputs to each receptor type. Additionally, GABAC receptor-mediated currents, activated electrically or by direct GABA application, have a slower time course than GABAA receptor-mediated currents (Amin & Weiss, 1994; Feigenspan & Bormann, 1994; Lukasiewicz & Shields, 1998). Although it is not yet known if these kinetic differences affect light-evoked signalling, it is possible that different amacrine cells may preferentially contact GABAA or GABAC receptor dominant synapses, suggesting that different types of amacrine cells could mediate slow and fast inhibition. Additionally, depending on the time constant of the rod bipolar cell membrane, slow currents might be more effective at producing changes in membrane voltage. A similar scheme of slow, tonic, currents having a larger impact than phasic currents was suggested in hippocampus (Nusser & Mody, 2002).
Different GABAergic and glycinergic amacrine cells also mediate distinct spatial signalling. In mammalian retina, GABAergic amacrine cells have processes with a wide lateral extent (Pourcho & Goebel, 1983) whereas glycinergic amacrine cells have more narrow-field processes (Vaney, 1990; Menger et al. 1998). In cat alpha ganglion cells, presynaptic glycinergic inhibition is mediated by narrow-field amacrine cells and GABAergic inhibition is mediated by wide-field amacrine cells (O'Brien et al. 2003), suggesting different functional roles for GABAergic and glycinergic inhibition in the retina. Narrow-field glycinergic cells may be more sensitive to local illumination changes or transmit information vertically through the retina, while the wide-field GABAergic amacrine cells may be more sensitive to changes in global illumination and transmit information laterally through the retina.
In addition to modulation of inhibitory timing, or spatial representation, our findings suggest that modulating the relative proportions of inhibitory inputs mediated by GABAA, GABAC and glycine receptors could adjust rod bipolar cell output. Currents mediated by these inhibitory neurotransmitter receptors in the retina have been shown to be changed by several neuromodulators and intracellular signalling cascades. Retinal glycine receptors are modulated by Zn2+ (Han & Wu, 1999), protein kinase A and protein kinase C (PKC) (Han & Slaughter, 1998), and GABAA receptors by Zn2+ (Li & Yang, 1999) and PKC (Gillette & Dacheux, 1996). Retinal GABAC receptor currents are modulated by dopamine (Wellis & Werblin, 1995) and mGluR1 agonists (Euler & Wässle, 1998), signals of important physiological relevance in the retina.
Our results suggest that modulating different receptor types that mediate inhibition at rod bipolar cell axon terminals could be an important mechanism in fine-tuning rod pathway signalling. Under our experimental conditions, GABAC receptor-mediated inhibition was dominant. Since GABAC receptors are more sensitive to GABA (Amin & Weiss, 1994; Feigenspan & Bormann, 1994), this could match well with the high sensitivity rod pathway during dark-adapted conditions. It is possible that under different conditions the relative roles of these pathways may be altered by activation of the previously mentioned modulatory pathways.
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). C, photomicrograph of a rod bipolar cell, filled with Lucifer yellow. The scale bar is 10 µM.
