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Departments of
1 Ophthalmology & Visual Science
3 Pharmacology & Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198–5540, USA
2 Department of Biology, Courant Institute of Mathematical Sciences and Center for Neural Science, New York University, New York, NY 10012, USA
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Abstract |
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(Received 11 August 2005;
accepted after revision 11 October 2005;
first published online 13 October 2005)
Corresponding author W. B. Thoreson: Department of Ophthalmology, University of Nebraska Medical Center, Durham Research Center, Omaha, NE 68198–5840, USA. Email: wbthores{at}unmc.edu
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Introduction |
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OFF bipolar and horizontal cells in amphibian and mammalian retina possess only non-NMDA ionotropic glutamate receptors (reviewed by Thoreson & Witkovsky, 1999). There are striking kinetic differences between KA- and AMPA-type non-NMDA receptors: AMPA receptors desensitize nearly tenfold faster than KA receptors (Dingledine et al. 1999) and native KA receptors exhibit even slower kinetics than expressed KA receptors (Kidd & Isaac, 2001). The cell-specific expression of different AMPA and KA receptors with different desensitization properties have been shown to shape the postsynaptic currents in different classes of cone-driven OFF bipolar cells from the ground squirrel retina (DeVries, 2000). We hypothesized that there may be similar differences at rod and cone synapses, but using selective pharmacological agents we found that glutamate receptors at both rod and cone synapses in the salamander retina are largely of the AMPA type. We also found no appreciable differences in the properties of spontaneous mEPSCs derived from rods and cones in any individual OFF bipolar or horizontal cell, although there were differences between cells. These results suggest that glutamate receptor properties do not contribute significantly to promoting faster transmission in the cone pathway.
To predict the impact of rod–cone differences in release kinetics, we need an appropriate model relating presynaptic release kinetics to the postsynaptic response. We hypothesized that the photoreceptor synapse adheres to the quantal hypothesis of exocytosis developed by del Castillo & Katz, 1954). According to this hypothesis, the EPSC derives from a linear superposition of unitary postsynaptic current events (mEPSCs) in a single-compartment (isopotential), voltage-clamped postsynaptic neurone. Although the quantal hypothesis can adequately account for postsynaptic responses at many CNS synapses (Redman, 1990; Sakaba et al. 2002), the graded response properties of photoreceptors, their ability to release glutamate for an indefinite period of time, the structure of the presynaptic ribbon with its numerous tethered vesicles, and the finding that pre- and postsynaptic processes are not in intimate association as in typical CNS synapses have prompted the suggestion that release from photoreceptor synapses may differ from that at conventional CNS synapses. Thus, it has been proposed that rather than being determined by the statistically independent actions of individual vesicles, the postsynaptic responses of second-order retinal neurones may instead reflect changes in the spatially integrated level of glutamate in the cleft determined by a balance between the release and re-uptake of glutamate (Gaal et al. 1998; Roska et al. 1998). To establish whether use of a quantal release model was appropriate, we analysed whether mEPSCs in OFF bipolar and horizontal cells exhibit statistical properties consistent with individual, independently released quanta. We then tested whether it was possible to reconstruct the EPSC by convolving the mEPSC waveform with the synaptic transmitter release rate function. The results show that convolution of the waveforms of spontaneously occurring mEPSCs with the empirically determined synaptic transmitter release rate function closely matched the EPSC waveforms evoked by depolarizing steps applied to cones. Convolution of mEPSCs with rod release functions also produced a good match to the EPSCs in some rod-driven neurones but the actual EPSCs were slower than those predicted by the convolution in a number of other rod-driven cells. We present evidence that this difference is partly explained by rod–rod coupling. The ability of the convolution to reproduce EPSC waveforms supports the quantal hypothesis at the photoreceptor synapse. Results of convolution experiments also indicate that rod–cone differences in synaptic release kinetics are a primary factor in shaping rod–cone differences in EPSC waveforms and are thus also likely to be a major factor in producing the faster cone synaptic impulse response.
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Methods |
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Aquatic tiger salamanders (Ambystoma tigrinum) 15–25 cm in length were handled humanely according to protocols approved by the Institutional Animal Care and Use Committee at the University of Nebraska Medical Center. The salamander was decapitated with heavy shears and the brain and spinal cord were rapidly pithed. After enucleation, the anterior segment of the eye was removed. The resulting eyecup was cut into thirds and a section was placed vitreal side down on a piece of filter paper (2 mm x 5 mm, Type AAWP, 0.8 µm pores, Millipore). After the retina adhered to the filter paper, the retina was isolated under chilled amphibian superfusate (for composition, see below). The retina and filter paper were cut into 125 µm slices using a razor blade (no. 121-6, Ted Pella Inc., Redding, CA, USA) tissue chopper (Stoelting, Wood Dale, IL, USA). Retinal slices were rotated 90 deg to permit viewing of the retinal layers when placed under a water immersion objective (40 x, 0.7 NA) and viewed on an upright fixed stage microscope (Olympus BHWI). For experiments examining possible rod–cone differences in mEPSC characteristics, dissection and viewing of slices was performed under infrared illumination using infrared-sensitive cameras (Watec 502H, Rock House Products, New York, NY, USA) mounted on the dissecting and recording microscopes. To produce cone-dominated conditions, rods were desensitized by a steady 480 nm background light of 
1.0 x 103 photons s–1µm–2.
Solutions were applied by a single-pass, gravity-feed perfusion system, which delivered superfusate to the slice chamber at a rate of 1 ml min–1. The normal amphibian superfusate contained (mM): 111 NaCl, 2.5 KCl, 2 CaCl2, 0.5 MgCl2, 10 Hepes and 5 glucose (pH 7.8). Use of Hepes as a pH buffer limited effects of proton feedback (DeVries, 2001; Hosoi et al. 2005). The solution also contained 1 µM strychnine and 100 µM picrotoxin to block glycine and GABAA/C receptors, respectively. The osmolarity was measured with a vapour pressure osmometer (Wescor, Logan, UT, USA) and adjusted, if necessary, to 242 ± 5 mosmol l–1. Solutions were bubbled continuously with 100% O2.
Electrophysiology
Patch pipettes were pulled on a PP-830 vertical puller (Narishige USA, East Meadow, NY, USA) from borosilicate glass pipettes (1.2 mm O.D., 0.9 mm I.D., with internal filament, World Precision Instruments, Sarasota, FL, USA) and had tips of 1 µm O.D. with resistance values of 10–15 M
. Pipettes were filled with a solution containing (mM): 48 caesium gluconate, 42 CsCl, 9.4 TEACl, 1.9 MgCl2, 9.4 MgATP, 0.5 GTP, 5 EGTA and 32.9 Hepes (pH 7.2). The osmolarity was adjusted, if necessary, to 242 ± 5 mosmol l–1.
For paired recordings, rods or cones were voltage clamped simultaneously with adjacent postsynaptic neurones using a Multiclamp patch-clamp amplifier (Axon Instruments Inc.). Both recording pipettes were positioned with Huxley-Wall micromanipulators (Sutter Instruments, Novato, CA, USA) and visualized with a video camera (Watec 502H) mounted on the microscope (Olympus BHWI). Currents were acquired using a Digidata 1322 interface and pCLAMP 8.1 software (Axon Instruments). Acceptable access resistances for voltage clamp recordings were < 50 M. Photoreceptors were voltage clamped at –70 mV, bipolar cells at –50 mV, and horizontal cells at –40 mV. In most cases, we stimulated the photoreceptor with a step from –70 to –10 mV to maximally stimulate calcium currents (ICa) and thereby evoke large and reproducible EPSCs. Stable recordings from synaptically coupled pairs of cells of > 15 min were needed for full washout and recovery from drug effects. Paired recordings were maintained for as long as 3 h.
Rods and cones were identified by their characteristic outer segments. Horizontal and OFF bipolar cells were identified by their response characteristics (Thoreson et al. 1997) and appearance after staining with Lucifer Yellow (2 mg ml–1) or AlexaFluor 568 (Molecular Probes; 0.2 mg ml–1). Rod input resistance averaged 320 ± 14 M and charging curves were fitted by single exponentials averaging 1.1 ± 0.1 ms (n= 13). Cone input resistance averaged 208 ± 11 M and charging curves were fitted by single exponentials averaging 2.6 ± 0.2 ms (n= 22). Input resistance of OFF bipolar cells averaged 1.5 ± 0.4 G and the charging curves could typically be fitted by single exponentials averaging 0.9 ± 0.1 ms (n= 21). Input resistance of horizontal cells averaged 747 ± 364 M and the charging curves could typically be fitted by single exponentials averaging 1.7 ± 0.4 ms (n= 13). The high input resistance and good fit by single exponential charging curves suggests that the horizontal cells were largely uncoupled from their neighbours in the retinal slice preparations used for these studies.
Chemicals were dissolved in amphibian superfusate except for GYKI 52466 and cyclothiazide, which were prepared in 10 000 x stock solutions of DMSO. Solvent control experiments with 1 : 1000 dilution of DMSO showed no effect on EPSCs. SYM 2081, NS 102, GYKI52466 and NBQX were obtained from Tocris Cookson (Ellisville, MO, USA). Unless otherwise stated, all other chemicals were obtained from Sigma-Aldrich.
Analysis
The mEPSC averaged from a series of individual mEPSCs was fitted by the best-fitting scale factor times the function: y=e(–t/
1)+be(–t/2)– (1 +b)e(–t/3). For each set of time constants and coefficient b, the best-fitting scale factor is given by the dot product of the vector of experimental response values measured at discrete times and the function y evaluated at the same times and normalized to have unit length. The ‘best-fit’ values of the free parameters (b, 1, 2 and 3) were determined by minimizing the sum of the square differences using Matlab (Mathworks) programs. The resulting fitted function for the mEPSC waveform was then convolved with empirically derived synaptic transmitter release rate functions. Release kinetics were obtained in a previous study by measuring exocytotic capacitance changes evoked by test pulses of varying duration (Rabl et al. 2005). For that study, capacitance (C)–duration plots were fitted with a dual exponential function: 
C=A1(1 – exp(–t/1)) +A2(1 – exp(–t/2)). The parameters that provided the best fits to the capacitance–duration relations were: for cones, 1= 2.7 ms, Amplitude1 (A1) = 306 fF, 2= 1.4 s, Amplitude2 (A2) = 474 fF; and for isolated rods, 1= 87 ms, A1= 132 fF, 2= 14 s, A2= 1170 fF (Rabl et al. 2005). In our model, the rate of release was taken to be proportional to the derivative of the capacitance–duration function.
The criterion for statistical significance in Student's t tests (GraphPad Prism 4.0) was chosen to be P < 0.05. Variability is reported as ±S.E.M.
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Results |
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Application of depolarizing steps (–70 to –10 mV) to a presynaptic cone or rod evoked sustained inward ICa that activated with a similar time course in both cell types, consistent with results of Rabl et al. (2005). Despite the sustained nature of ICa in rods and cones, the EPSCs recorded simultaneously in postsynaptic OFF bipolar or horizontal cells were transient (Fig. 1). The ability of a sustained ICa to produce transient EPSCs is similar to results at the rod bipolar to AII amacrine cell synapse (Singer & Diamond, 2003).
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By evoking EPSCs in a single second-order neurone with presynaptic stimulation of first a cone and then a rod (or vice versa), we tested whether rod–cone differences in EPSC kinetics involved a general property of the postsynaptic neurone or a specific property of rod and cone synapses. In every case where we successfully recorded from both a rod and a cone while maintaining the postsynaptic recording from an OFF bipolar or horizontal cell (n= 11), the EPSC evoked by presynaptic stimulation (200 ms step, –70 to –10 mV) of the cone was much more transient than the EPSC evoked in the same postsynaptic neurone by stimulation of the rod (Fig. 2). Overlaying the EPSCs evoked by a rod (black trace) and cone (grey trace) in the same OFF bipolar cell also reveals that rod-evoked EPSCs attain their peak later than cone-evoked EPSCs (Fig. 2) although the latency to the beginning of the EPSC was the same for both rod- and cone-evoked EPSCs. The kinetic differences between the EPSCs evoked by depolarizing steps applied to rods versus cones are thus due to properties of the individual synapses made by rods and cones onto postsynaptic cells.
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on= 1.91 ± 0.15 ms, n= 58) exhibited a significantly faster rise time than rod-driven EPSCs (on= 7.51 ± 0.62 ms, n= 38). To illustrate this difference, rod- and cone-driven EPSCs with rise times near the average are overlaid in Fig. 3B. Time constants for the decay of the EPSC were also significantly faster in cone-driven cells (7.97 ± 0.97 ms, n= 52) than rod-driven cells (57.88 ± 5.94 ms, n= 32; Fig. 3C). Representative rod- and cone-driven EPSCs with decay times near the average are overlaid in Fig. 3D. Limiting analysis to the 52 cone-driven cells and 29 rod-driven cells in which onset and decay could both be fitted with single exponentials yielded time constants for onset (cones, 1.70 ± 0.13 ms, P= 0.29; rods, 6.84 ± 1.18 ms, P= 0.37) and decay (cones, 6.84 ± 1.05 ms, P= 0.73; rods, 46.4 ± 4.64 ms, P= 0.14) that were not significantly different from the larger sample shown in Fig. 3A.
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In most experiments, large depolarizing test steps from –70 to –10 mV were used to evoke large, reproducible EPSCs. Weaker depolarizing steps to –30 mV, near the normal photoreceptor dark resting potential, evoked slower EPSCs than were evoked by steps to –10 mV (Fig. 3). Nevertheless, cone-driven EPSCs evoked by steps to –30 mV continued to show faster onset (5.19 ± 1.00 ms, n= 19) and decay (12.28 ± 2.46 ms, n= 14) kinetics than rod-driven EPSCs (on= 11.37 ± 1.85 ms, n= 15, decay= 62.15 ± 5.52 ms, n= 13) evoked by the same step (Fig. 3).
Effects of cyclothiazide
OFF bipolar and horizontal cells of amphibian retina possess non-NMDA ionotropic glutamate receptors (reviewed by Thoreson & Witkovsky, 1999). The major subtypes of non-NMDA receptors desensitize at very different rates with AMPA receptors desensitizing nearly 10 times faster than KA receptors (Dingledine et al. 1999). We therefore examined the effects of drugs that act selectively on KA or AMPA receptors. To test the pharmacology of rod and cone contacts, we obtained data from rods or cones synaptically connected to either horizontal cells or OFF bipolar cells. Cells were filled with a fluorescent dye to permit anatomical characterization at the end of the recording. Confocal stacks of fluorescently stained pairs of cells are shown in Fig. 4. We grouped the data into four sets for analysis: (1) cone-driven horizontal cells (e.g. Fig. 4A); (2) cone-driven OFF bipolar cells (e.g. Fig. 4B); (3) rod-driven horizontal cells (e.g. Fig. 4C); and (4) rod-driven OFF bipolar cells (e.g. Fig. 4D).
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Agonist/antagonist pharmacology
The results with CTZ suggest a significant contribution from AMPA receptors in rod- and cone-driven horizontal and OFF bipolar cells but do not preclude the simultaneous presence of KA receptors. Furthermore, although we found that CTZ did not affect photoreceptor ICa (not shown), we could not exclude the possibility that CTZ may act presynaptically (Diamond & Jahr, 1995; Bellingham & Walmsley, 1999). We therefore used AMPA- and KA-selective agonists and antagonists to further test the possibility that AMPA receptors play a dominant role in both rod- and cone-driven EPSCs.
EPSCs in rod-driven OFF bipolar cells (n= 7, Fig. 5A) and rod-driven horizontal cells (n= 7, Fig. 6A) were strongly reduced or completely blocked by the selective AMPA antagonist GYKI 52466 (3–10 µM). In the examples shown in the figures, washout was begun before GYKI 52466 had exerted its full blocking effect. Full block was consistently obtained when GYKI 52466 application was maintained for a longer time but full recovery of the EPSC during the recording was not obtained in these cases. EPSCs in rod-driven horizontal cells were also inhibited by the competitive AMPA antagonist NBQX (3–10 µM, n= 9, Fig. 6B).
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Consistent with results from rod-driven cells, the selective AMPA antagonist GYKI 52466 (3–10 µM) strongly inhibited EPSCs in 7 of 8 cone-driven OFF bipolar cells (Fig. 7A) and 4 of 4 cone-driven horizontal cells (Fig. 8A). The failure of GYKI 52466 to suppress EPSCs in one cone-driven OFF bipolar cell suggests the possible presence of KA receptors but the recording was lost before KA-selective drugs could be tested. SYM 2081 (10–30 µM) and NS 102 (20 µM) had no effect on EPSCs in cone-driven OFF bipolar cells (Fig. 7; SYM 2081, n= 3; NS 102, n= 4) or cone-driven horizontal cells (Fig. 8; SYM 2081, n= 4; NS 102, n= 7). Although it did not affect EPSC amplitude, SYM 2081 produced a small inward current in the horizontal cell shown in Fig. 8 suggesting the possibility of some KA receptors located elsewhere on the cell. Taken together, the experiments described above indicate that in most OFF bipolar and horizontal cells, rods and cones both contact pharmacologically similar AMPA-type glutamate receptors.
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We examined the properties of spontaneously occurring mEPSCs in OFF bipolar and horizontal cells in order to determine whether they represent independent, unitary events appropriate for convolution with the release function. Spontaneous mEPSCs were consistently observed while recording from OFF bipolar cells; these results are in agreement with earlier studies (Maple et al. 1994; Kawai, 1999; Wu et al. 2000). Horizontal cells were often uncoupled in the slice as evidenced by their high input resistance and, consistent with other studies (Hirasawa et al. 2001), uncoupled horizontal cells also exhibited mEPSCs. Examples of mEPSCs recorded from OFF bipolar cells at holding potentials of –50 and –90 mV are shown in Fig. 9A and B, respectively. These spontaneous synaptic currents were considered to be mEPSCs since they were recorded in the presence of picrotoxin (100 µM) and strychnine (1 µM) used to block GABAA/C receptors and glycine receptors, respectively. Furthermore, spontaneous synaptic currents recorded under these conditions were blocked by NBQX and GYKI 52466 (e.g. Figs 5–7) and reversed near 0 mV (not shown) indicating that they were glutamatergic.
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Most OFF bipolar and horizontal cells in the salamander retina receive inputs from both rods and cones (Hensley et al. 1993; Thoreson et al. 2003). Thus, spontaneous mEPSCs in these cells can arise from vesicles released by both types of photoreceptor cells. Maple et al. (1999) reported that the single channel conductance of AMPA receptors in cone-driven OFF bipolar cells was less than half that of AMPA receptors in rod-driven OFF bipolar cells. Assuming that vesicles in rod and cone terminals activate the same number of glutamate receptors, then similar differences between glutamate receptors at rod and cone synapses in individual neurones should result in a bimodal distribution in the amplitude of mEPSCs. However, although slightly skewed as described above, amplitude distributions of mEPSCs were largely unimodal (Fig. 9A and B). The mean amplitude of mEPSCs recorded at a holding potential of –90 mV averaged 6.5 ± 1.6 pA (n= 10), ranging from 3.7 to 8.8 pA. The wide range of mEPSC amplitudes between cells suggests that although amplitude distributions within individual cells were unimodal, different cells (e.g. rod- versus cone-driven OFF bipolar cells) may possess AMPA receptors with differing properties.
Like amplitude histograms, half-amplitude time width distributions were consistently unimodal. They were well fitted by a single Gaussian function (Fig. 9C) suggesting little kinetic difference between mEPSCs arising from rod and cone inputs. Inter-event interval histograms were fitted by single exponential functions consistent with a Poisson release process involving the random release of vesicles from a large releasable pool (Fig. 9D; n= 5).
As a more direct test for possible differences between mEPSCs arising from rods and cones, we recorded spontaneous mEPSCs from dark-adapted OFF bipolar and horizontal cells and then reduced rod output by applying a 480 nm adapting light. Dark-adapted responses of second-order neurones from slices prepared under infrared light are rod dominated (Thoreson et al. 2003). Application of a 480 nm background light (1 x 106 photons s–1µm–2) strongly hyperpolarizes rods and blocks their light responses but only slightly diminishes cone responses (Thoreson et al. 2003). Thus, use of a 480 nm background light should preferentially reduce the spontaneous release of vesicles from rods. Consistent with reduced synaptic output, the frequency of spontaneous mEPSCs was reduced from 83.9 ± 11.1 to 60.6 ± 10.6 mEPSC s–1 by the adapting light (P < 0.0001, paired t test, n= 6). As illustrated by the cumulative histograms in Fig. 10, reducing rod output with the adapting light altered neither the amplitude nor half-amplitude time width of mEPSCs. Similar results were observed in all nine cells that were tested. The amplitude and half-amplitude time width of mEPSCs were also unaltered by using a background light that was 2.8 log units more intense. These results indicate that mEPSCs arising from rods and cones are similar suggesting that vesicles derived from both cell types release similar quantities of glutamate and act on postsynaptic glutamate receptors with similar properties.
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Cones exhibit an initial component of exocytosis that is more than tenfold faster than the initial component of exocytosis from rods (Rabl et al. 2005). The quantal hypothesis predicts that the EPSC can be constructed from a linear superposition of unitary postsynaptic current events (mEPSCs) in a single-compartment (isopotential), voltage-clamped postsynaptic neurone, as long as the glutamate receptors are not saturated. To test the quantal hypothesis and assess the impact of rod–cone differences in release, we computed theoretical EPSCs by convolving the mEPSC waveform with the synaptic transmitter release rate functions for rods and cones.
Experiments described above indicated that mEPSCs in OFF bipolar and horizontal cells are unitary quantal events released in a statistically independent manner. Furthermore, the properties of mEPSCs in a given cell exhibited a single unimodal distribution and there was no evidence for differences in mEPSCs derived from rods and cones. For these reasons, we concluded that mEPSCs derived from any individual synaptic input are similar to the overall population of spontaneously occurring mEPSCs in a given cell. We therefore created an mEPSC waveform for each cell by averaging together a number of individual mEPSCs (insets in Fig. 11). To produce a smooth function describing the mEPSC waveform, the average mEPSC was fitted with a three-exponential function as described in Methods.
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1= 3 ms, 2= 1.4 s; Rabl et al. 2005). The cumulative release function, from which the release rate function was derived, was measured previously using capacitance measurement techniques (Rabl et al. 2005). Despite the fact that the release function was measured in a different sample of cells using different techniques, the EPSC waveform predicted by the convolution provided a remarkably good match to the observed EPSC (Fig. 11A). Good matches were obtained for all seven cone-driven pairs of cells that were analysed (4 cone–OFF bipolar cell pair, 3 cone–horizontal cell pairs). To illustrate the overall fit to this sample of cells, we convolved the mEPSC averaged among these seven postsynaptic neurones (after normalizing mEPSC amplitude between cells). As shown in Fig. 11B, convolution of the average mEPSC produced a waveform that closely predicted the actual EPSC waveform averaged from this same sample of cone-driven neurones. The average EPSC waveform exhibited onset and decay time constants (on= 1.55 ms, decay= 6.11 ms) similar to the much larger group of cells analysed for Fig. 3 suggesting that this sample was representative of the overall population. By contrast with convolution using the cone release function, convolving the average mEPSC from these cone-driven cells with the rod release function produced a substantially slower waveform (Fig. 11C). The ability of linear convolution to predict the EPSC waveform in this sample of cells provides support for the quantal hypothesis. We used the same approach to analyse rod-driven EPSCs. Figure 11D shows the EPSC and average mEPSC (inset) obtained from a horizontal cell while simultaneously recording from a presynaptic rod. For the rod-driven cells, we used an exponential release function with time constants of 87 ms and 14 s to convolve the mEPSC waveform (Rabl et al. 2005). Note the fivefold slower timescale of the rod-driven responses in Fig. 11D–F compared with the cone-driven responses in Fig. 11A–C. In the example shown in Fig. 11D, the convolution produced a good match to the overall envelope of the EPSC waveform although it did not reproduce the small reduction in current that immediately followed the initial peak. Prominent secondary peaks similar to Fig. 11D were observed in other rod-driven EPSCs (e.g. Figures 4D and 12B); experiments described in the following section (Fig. 12) suggest that this delayed secondary component to the EPSC involves synaptic output from coupled rods. The failure of the convolution to capture the detailed structure of the EPSC was not unexpected, since the release function was obtained from capacitance measurements made at a handful of discrete time points using a large sample of cells which would obscure detailed differences in release kinetics among individual cells.
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on= 5.8 ms, decay= 66.7 ms) similar to the much larger group of cells analysed for Fig. 3 suggesting that this sample is representative of the larger population. The peak and the rise time of the average EPSC were somewhat slower than the waveform predicted by the convolution. Nonetheless, convolution of the mEPSC with the rod release function produced a far better match to the rod-driven EPSC (Fig. 11E) than to the cone-driven EPSC (Fig. 11C). Conversely, convolution with the cone release function produced a much better match to the cone-driven EPSC (Fig. 11B) than to the rod-driven EPSC (Fig. 11F). These results suggest that the slower kinetics of release from rod terminals contribute significantly to producing the slower rod-driven EPSCs. Impact of rod–rod coupling
The presence of strong rod–rod coupling in the amphibian retina (Attwell et al. 1984; Zhang & Wu, 2005) raises the possibility that rod-driven EPSCs may include a component of exocytosis arising from the stimulation of adjacent rods by current spread through gap junctions. We therefore tested effects of gap junction blockers (Rozental et al. 2001) on the EPSC. Using paired rod–rod recordings, we established that carbenoxolone (0.2 mM) reversibly inhibited the gap junctional conductance between rods (Fig. 12A, n= 7). Using paired pre- and postsynaptic recordings, we then found that carbenoxolone reversibly inhibited later portions of the rod-driven EPSC in horizontal and OFF bipolar cells (Fig. 12B, n= 5). Photoreceptor Ca2+ channels can be inhibited by carbenoxolone (Vessey et al. 2004) and this would contribute to a reduction in the EPSC. However, in an experiment in which we interleaved measurements of ICa with ramp voltage protocols between tests of the EPSC with depolarizing steps, we found that the reduction in the late component of the EPSC preceded any significant change in ICa. As a control for non-specific effects on synaptic transmission, we tested the effects of carbenoxolone on synaptic transmission from cones to horizontal cells. The onset and decay kinetics of cone-driven EPSCs in horizontal cells were not significantly altered by application of carbenoxolone (0.2 mM; n= 4 pairs; on, P= 0.57; decay, P= 0.24). We also tested a second, structurally dissimilar gap junction blocker, heptanol (2 mM; Rozental et al. 2001), and found that it inhibited the gap junctional conductance between rods and produced a similar preferential reduction in the late component of the EPSC (n= 2, not shown). These experiments suggest that gap junctional coupling between rods leads to a delayed component in the EPSC, consistent with the low-pass filtering expected from rod–rod coupling (Zhang & Wu, 2005).
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Discussion |
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100 ms to achieve half-maximal desensitization suggesting that desensitization has little impact on the decay of depolarization-evoked EPSCs at cone- or rod-driven synapses, which exhibited decay time constants of 8 ms and 60 ms, respectively. Maple et al. (1999) also reported that the single channel conductance of AMPA receptors in cone-dominated cells was 43% of that in rod-dominated cells. Assuming that vesicles released by rods and cones activate the same number of glutamate receptors, the presence of a similar difference in conductance at the synapses made by rods and cones on individual cells should produce a bimodal mEPSC amplitude histogram. However, we found that amplitude distributions were unimodal within any individual cell and there was no evidence for rod–cone differences in the amplitude of mEPSCs. Although we found a large variation in the mean amplitude of mEPSCs from cell to cell (with the smallest amplitude mEPSC being 42% of the largest) that is consistent with the possibility of differences in glutamate receptors between different cells (e.g. between rod- and cone-dominated cells), our results suggest there are no significant differences in the properties of glutamate receptors clustered at rod and cone synapses within any individual cell.
Kim & Miller (1991, 1993) found that concentrations of 0.5–1 mM kynurenic acid were required to block EPSCs in horizontal and OFF bipolar cells evoked by depolarizing steps applied to presynaptic cones whereas 
5 mM kynurenic acid was needed to block EPSCs in rod-driven horizontal and OFF bipolar cells. They proposed that these differences may reflect differences in glutamate receptors at rod and cone synapses, but subsequent studies showed that the effective concentration of the low affinity glutamate receptor antagonist, kynurenic acid, can be interpreted as reflecting the peak concentration of glutamate attained at the synapse (Clements, 1996). This interpretation takes advantage of the fact that the ratio between the binding rates of glutamate and the more rapidly dissociating antagonist is similar to the ratio between their concentrations (Clements, 1996). Thus, rather than implying pharmacologically distinct receptors, differences in the concentrations of kynurenic acid needed to block EPSCs evoked by strong depolarization of rods versus cones may instead suggest that postsynaptic glutamate receptors adjacent to cones are exposed to glutamate concentrations in excess of 0.5 mM and those adjacent to rods are exposed to concentrations exceeding 5 mM. Synaptic vesicles in rods and cones contain similar amounts of glutamate (Fig. 10), synaptic ribbons appear to tether the releasable pool of vesicles (von Gersdorff et al. 1996; Thoreson et al. 2004), and rods and cones have similar size ribbons (Pierantoni & McCann, 1981; Thoreson et al. 2004). Although cones exhibit a larger exocytotic capacitance response than rods (Rabl et al. 2005), it thus appears likely that rods and cones release a similar amount of glutamate at each ribbon; the larger total release from cones is probably due to a greater number of ribbons. Cones can also release glutamate more rapidly than rods (Rabl et al. 2005) but the amount reaching postsynaptic receptors depends on diffusional distance from the release site and efficiency of glutamate uptake, which may differ at rod and cone synapses. One cannot therefore readily predict the concentration of glutamate in the synaptic cleft of rod and cone photoreceptors in the amphibian retina. However, it is worth noting that these concentration values are consistent with results at other CNS synapses where the release of a vesicle causes a spatially restricted elevation of glutamate levels to concentrations of 1–5 mM (Clements, 1996).
Depolarization evokes a tenfold faster initial burst of exocytosis from cones than rods (Rabl et al. 2005). A major goal of the present study was to determine how these differences in release rate shape the postsynaptic responses of OFF bipolar and horizontal cells. To model the impact of release rates on the postsynaptic response, we assumed that the photoreceptor synapse adheres to the quantal hypothesis of exocytosis (del Castillo & Katz, 1954) and that the EPSC derives from a linear superposition of unitary postsynaptic current events (mEPSCs) in a single compartment (isopotential) postsynaptic neurone. The assumption of isopotentiality is supported by the finding that OFF bipolar cells exhibited single exponential charging curves consistent with single compartment structures. Horizontal cells in which mEPSCs were detected – and were thus presumably uncoupled from their neighbours – also exhibited single exponential charging curves. The assumption that mEPSCs represent unitary quantal events is supported by the unimodal distributions of amplitudes and half-amplitude time widths. Spontaneous mEPSCs detected in a postsynaptic neurone can derive from any one of a number of presynaptic photoreceptors. However, the unimodal nature of mEPSC distributions and the absence of any rod–cone differences in the properties of mEPSCs or glutamate receptor pharmacology suggests that in any given second-order neurone, the properties of the overall population of spontaneously occurring mEPSCs are representative of mEPSCs derived from any individual photoreceptor. To obtain the mEPSC waveform we therefore averaged a number of spontaneously occurring mEPSCs. The average mEPSC was fitted with a three exponential function to produce a smooth waveform. There was a slight deviation between the fitted waveform and the actual mEPSC during the initial rising phase. This deviation may contribute to deviation between the rising phase of the EPSC predicted from convolution and the actual EPSC. Nevertheless, convolution of the mEPSC waveform fit with the cone release function produced a remarkably good match to the actual cone-driven EPSCs. The ability to reconstruct the EPSC by linear convolution of individual mEPSCs supports the hypothesis that postsynaptic responses of OFF bipolar and horizontal cells arise from a linear summation of individual quantal release events. The success of the convolution implies that even at the highest release rates achieved at the peak of the EPSC there is little spatial or temporal overlap in the effects of individual quanta. These data argue against the alternative hypothesis that postsynaptic responses of second-order retinal neurones are not quantal in nature but instead reflect changes in the spatially integrated level of glutamate in the cleft (Gaal et al. 1998; Roska et al. 1998).
Convolution of mEPSCs with the rod release function also produced a good match in some rod-driven neurones, but the actual EPSC was slightly slower than the predicted EPSC in many cells. As described above for cone-driven EPSCs, deviations between the actual mEPSC and the fitted waveform may have contributed to the faster rise time of the predicted EPSC compared with the actual EPSC. Nevertheless, convolution with the rod release function produced a far better match to rod-driven EPSCs than convolution of the same mEPSC waveform with the cone release function, consistent with the hypothesis that presynaptic differences in the rates of release are a major factor in producing the dramatic kinetic differences between rod- and cone-driven EPSCs.
The presence of strong rod–rod coupling in the amphibian retina (Attwell et al. 1984; Zhang & Wu, 2005) also contributes to the slower decay of rod- versus cone-driven EPSCs. Inhibition of rod–rod coupling with carbenoxolone or heptanol preferentially reduced a delayed component to the EPSC in rod-driven cells consistent with the suggestion that the spread of depolarizing current through gap junctions stimulates glutamate release from neighbouring rods. It should be noted that the release function was measured in uncoupled cells (Rabl et al. 2005) and thus any contribution from coupling to the rod-driven EPSC would not be predicted by convolution with the release function.
In summary, the present results indicate that rod–cone differences in release kinetics play a major role in shaping the postsynaptic currents evoked by depolarizing steps applied to presynaptic photoreceptors. Faster cone-driven EPSCs (Fig. 3) and faster release kinetics (Rabl et al. 2005) are observed at membrane potentials near the dark resting potential suggesting that rod–cone differences in release kinetics are present under physiological conditions and thus may play a role in shaping postsynaptic responses to light. Schnapf & Copenhagen (1982) computed the synaptic impulse responses of rods and cones by measuring photoreceptor and horizontal cell responses to brief light flashes and found the cone synaptic impulse response to be 8–10 times faster than the synaptic impulse response of rods. These differences in synaptic kinetics roughly match differences in the kinetics of the light response of rods and cones (Baylor & Hodgkin, 1973; Pasino & Marchiafava, 1976; Schnapf & Copenhagen, 1982; Copenhagen et al. 1983). Horizontal and bipolar cells respond linearly to modulations of light intensity of low to intermediate contrast around a background light level (Tranchina et al. 1981; Burkhardt et al. 2004). In the linear regime of stimulus contrast, responses to incremental and decremental steps of light are mirror images of one another. This suggests that in the presence of a background light level, the processes responsible for a rapid increase in exocytosis from cones at light offset may also support rapid changes in synaptic output at light onset. Thus, in addition to promoting differences in the kinetics of depolarization-evoked EPSCs, rod–cone differences in release kinetics are also likely to be an important factor in producing the faster synaptic impulse response of cones.
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) was normalized to the amplitude of the event histogram and fitted with a single Gaussian curve (S.D.= 0.66 pA). B, spontaneous mEPSCs and amplitude histogram (•, 757 events) from a different OFF bipolar cell when Vh=–90 mV. The normalized mEPSC amplitude histogram was fitted with a gamma distribution (
