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The Journal of Neuroscience, July 9, 2003, 23(14):6063-6073

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Light-Evoked Excitatory and Inhibitory Synaptic Inputs to ON and OFF Ganglion Cells in the Mouse Retina

Ji-Jie Pang, Fan Gao, and Samuel M. Wu

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

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bipolar cell and amacrine cell synaptic inputs to ganglion cells (GCs) in dark-adapted mouse retinas were studied by recording the light-evoked excitatory cation current (I_C) and inhibitory chloride current (I_Cl) under voltage-clamp conditions, and the cell morphology was revealed by Lucifer yellow fluorescence with a confocal microscope. Three types of GCs were identified. (1) ONGCs exhibits no spike activity in darkness, increased spikes in light, sustained inward I_C, sustained outward I_Cl of varying amplitude, and large soma (20–25 µm in diameter) with-cell-like dendritic field 180–350 µm stratifying near 70% of the inner plexiform layer (IPL) depth. (2) Transient OFFGCs (tOFFGCs) exhibit no spike activity in darkness, transient increased spikes at light offset, small sustained outward I_C in light, a large transient inward I_C at light offset, a sustained outward I_Cl, and a morphology similar to the ONGCs except for that their dendrites stratified near 30% of the IPL depth. (3) Sustained OFFGCs exhibit maintained spike activity of 5–10 Hz in darkness, sustained decrease of spikes in light, sustained outward I_C, sustained outward I_Cl, and a morphology similar to the tOFFGCs. By comparing the response thresholds and dynamic ranges of GCs with those of the preganglion cells, our data suggest that the light responses of each type of GCs are mediated by different sets of bipolar cells and amacrine cells. This detailed physiological analysis complements the existing anatomical results and provides new insights on the functional roles of individual synapses in the inner mammalian retina.

Key words: ganglion cells; light responses; rod bipolar cells; M-cone bipolar cells; S-cone bipolar cells; AII amacrine cells; response threshold; dynamic range

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Parallel information processing is a fundamental principle for sensory signal encoding in the brain, and two of the most important and well described signaling pathways in the visual system are the ON and OFF channels (Hubel and Wiesel, 1968). Anatomical studies during the past 30 years have suggested that the ON and OFF synaptic pathways in all mammal retinas follow the same plan: cones make synapses on cone depolarizing (ON) and hyperpolarizing (OFF) bipolar cells (DBC_Cs and HBC_Cs), which synapse on the ON and OFF ganglion cells, respectively (Kolb and Famiglietti, 1974; Nelson et al., 1978, 1981). Rods make synaptic contacts with only one type of bipolar cell that depolarizes in response to light spots (DBC_R). DBC_Rs synapse on the AII amacrine cells that make electrical synapses with DBC_Cs and inhibitory chemical synapses (probably glycinergic) with HBC_Cs and OFF ganglion cells (Bolz et al., 1984; Pourcho and Owczarzak, 1991; Crooks and Kolb, 1992). According to this plan, HBCs do not receive inputs directly from rods, and ON and OFF ganglion cells do not receive inputs directly from rod bipolar cells but by the DBC_R–AII pathway (Kolb and Nelson, 1981, 1983; Wassle and Boycott, 1991). Evidence from recent studies, however, begins to challenge this view. In the rabbit retina, for example, when rod–DBC_R synapses are blocked by APB, rod inputs to OFF ganglion cells persist, indicative of an alternative rod–OFF ganglion cell synaptic pathway (DeVries and Baylor, 1995). Studies on normal and coneless transgenic mice indicate that rods make synaptic inputs directly to HBCs (Soucy et al., 1998; Tsukamoto et al., 2001). These results suggest that the physiological responses of ON and OFF ganglion cells in mammalian retinas may be mediated by a synaptic network more complex than the general plan set forth by previous anatomical studies. Additionally, it is not clear whether all anatomically identified synapses made on ganglion cells are functional and how effective each of the bipolar cell and amacrine cell synapses transfers light-evoked signals. Systematic physiological analysis of the synaptic inputs mediating light-evoked excitatory and inhibitory responses of ON and OFF ganglion cells is needed to resolve these outstanding questions.

Most ganglion cell light responses in mammalian retina have been recorded by single or arrays of extracellular electrodes (Kuffler, 1953; Meister et al., 1991). The advantage of these techniques is that they allow stable recordings for long periods of time with minimum electrode damage to the cells. The disadvantages of this approach, however, include the inability to measure the transmembrane potential, inability to separate the various synaptic components of light responses, and the inability to inject intracellular dyes to reveal the cell morphology. In this study, we used the whole-cell voltage-clamp technique to record light-evoked responses from the ON and OFF ganglion cells in the dark-adapted flat-mount mouse retina. We chose the mouse retina because physiological results obtained in wild-type mice can later be correlated with findings in genetically manipulated mice. We chose ganglion cells (GCs) because they can be clearly distinguished from other ganglion cells by their distinct morphological signature (Sun et al., 2002), and their large somas are suitable for stable patch-clamp recordings. Moreover, the cellular and synaptic inputs to these cells have been well characterized at the ultrastructural level (Freed and Sterling, 1988; Vardi et al., 1989), and thus physiological findings can be readily correlated with anatomical observations.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Experimental approach. Our study constitutes the first voltage-clamp analysis of ganglion cell light responses in the mouse retina, and the major advantage of this approach is that excitatory and inhibitory current responses can be separated by holding the membrane potential near chloride and cation reversal potentials, respectively. Thus, the bipolar cell and amacrine cell contributions to the light responses of the ganglion cells (i.e., cation and chloride currents, or I_C and I_Cl, respectively) can be differentially recorded. Because mouse rods are 2–4 log units more sensitive to green light than cones (Lyubarsky et al., 1999), we use the response threshold to 500 nm lights to estimate the relative rod–cone contributions to I_C and I_Cl in each ganglion cell and thereby determine whether the light responses of the cell are driven by rod- or cone-dominated bipolar–amacrine cells. Another advantage of this approach is that the three-dimensional morphology of the cell can be easily revealed by Lucifer yellow filling with the recording electrode (assisted by a confocal microscope). This allows us to characterize the morphology of each recorded cell and to compare it with results of previous anatomical studies. It also allows us to exclude other types of ganglion cells and displaced amacrine cells [the latter accounts for 60% of the somas in the mouse ganglion cell layer (Williams et al., 1996; Jeon et al., 1998) and can be easily recognized by the lack of axons]. Additionally, spontaneous and light-evoked spike activities can be conveniently recorded with the patch electrodes in the "loose-patch" mode, permitting us to compare voltage-clamp current responses with the physiological responses of the unclamped cells.

Preparations and light stimulation. The mouse strain used in this study was C57BL/6J from The Jackson Laboratory (Bar Harbor, ME). All animals were handled in accordance with the policies on the treatment of laboratory animals of Baylor College of Medicine. Mice were dark adapted for 1–2 hr before the experiment. To maintain the retina in the fully dark-adapted state, all additional procedures were performed under infrared illumination with dual-unit Nitemare (BE Meyers, Redmond, WA) infrared scopes. Animals were killed by a lethal injection of ketamine plus xylazine plus acepromazine (0.1 ml, 100 mg/ml), and the eyes were immediately enucleated and placed in oxygenated Ames' medium (Sigma, St. Louis, MO) at room temperature. Dissection and preparation of flat-mount retinas followed essentially the procedures described by others (Werblin, 1978; Wu, 1987). Oxygenated Ames' solution (adjusted at pH 7.3) was introduced continuously to the recording chamber, and the medium was maintained at 34°C by a temperature control unit (TC 324B; Warner Instruments, Hamden, CT). All pharmacological agents were dissolved in Ames' medium.

A photostimulator was used to deliver light spots (diameter of 600–1200 µm) to the retina via the epi-illuminator of the microscope. The intensity of unattenuated (log I = 0) 500 nm light was 1.4 x 10⁶ photons µm^-2 sec^-1. The number of photoisomerizations per rod per second (Rh^*rod^-1sec^-1) was calculated by using a rod cross section of 0.5 µm^-2 (Howes et al., 2002) and a rod integration time of 0.4 sec (Baylor, 1987). The peak amplitude of light-evoked current responses was plotted against light stimulus intensity, and data points were fitted by the Hill equation: R/R_max = I^N/(I^N + ^N) = 0.5[1 + tanh 1.15N(Log I - Log )], where R is the current response amplitude, R_max is the maximum response amplitude, is the light intensity that elicits a half-maximal response, N is the Hill coefficient, tanh is the hyperbolic tangent function, and Log is the logarithmic function of base 10. In this article, we used the R - Log I plot for our analysis (the right-hand term of the above equation), and, for such plots, the light intensity span [dynamic range (DR), range of intensity that elicits responses between 0.05 and 0.95% of R_max] of a cell equals to 2.56/N (Thibos and Werblin, 1978).

Voltage-clamp recordings. Voltage-clamp recordings were made with an Axopatch 200A amplifier connected to a DigiData 1200 interface and pClamp 6.1 software (Axon Instruments, Foster City, CA). Spike activities were recorded extracellularly with patch electrodes made with Narishige (Tokyo, Japan) or Sutter Instruments (Novato, CA) patch electrode pullers that were of 1–2 M tip resistance when filled with Ames' medium in the loose-patch configuration. Whole-cell voltage-clamp recordings were made with patch electrodes of 5–7M tip resistance when filled with internal solution containing the following (in mM): 118 Cs methanesulfonate, 12 CsCl, 5 EGTA, 0.5 CaCl₂, 4 ATP, 0.3 GTP, 10 Tris, and 0.8 Lucifer yellow, adjusted to pH 7.2 with CsOH. The chloride equilibrium potential, E_Cl, with this internal solution was approximately -60 mV. Estimates of the liquid junction potential at the tip of the patch electrode before seal formation varied from -9.2 to -9.6 mV (Pang et al., 2002). For simplicity, we corrected all holding potentials by 10 mV. To determine the dark membrane potentials of ganglion cells, we measured the zero-current potentials of 12 ganglion cells (including all three cell types described in this paper) with patch electrodes filled with Cs internal solution (above) and with potassium internal solution (Berntson and Taylor, 2000) and found that the zero-current potentials with K⁺ were consistently 13–16 mV more hyperpolarized than with Cs⁺. For cells recorded with only Cs⁺, we corrected zero-current potential measured in darkness (dark membrane potential) by 15 mV. Because ganglion cells have relatively large dendritic fields, it is possible that we were unable to control the voltage of the fine dendrites. We therefore selected cells with higher input resistance (>500 M) when whole-cell recording was made and discarded any cell that showed unclamped spikes (typical from cells whose voltage at remote dendrites are not controlled).

Visualization of cell morphology. Three-dimensional cell morphology was visualized in flat-mount retinas through the use of Lucifer yellow fluorescence with a confocal microscope (model 510; Zeiss, Oberkochen, Germany). For vertical sections, retinas were subsequently fixed in fresh 4% paraformaldehyde with 0.05% gluderaldehyde for 15 min, transferred to 4% paraformaldehyde for another 2 hr, and then sectioned with a vibratome. Images were acquired with a 40x water immersion objective (numerical aperture, 1.20), using 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 (Adobe Systems, San Jose, CA) to improve the signal-to-noise ratio. Because signal intensity values were typically enhanced during processing to improve visibility of smaller processes, the cell bodies and larger processes of some cells appear saturated attributable to their larger volume of fluorophore. Although background images of the retinal sections were acquired simultaneously with the fluorescent cells, they were imaged by using transmitted light. The level at which dendritic processes stratified in the inner plexiform layer (IPL) was characterized in retinal vertical sections by the distance from the processes to the distal margin (0%) of the IPL. ganglion cells were identified by their three-dimensional morphology revealed by Lucifer yellow fluorescent images in retinal flat mounts and vertical sections: a large soma (20–25 µm in diameter) with an axon (therefore, we knew they were ganglion cells instead of displaced amacrine cells) and several stout primary dendrites emerging radially, and higher-order dendrites with a beaded appearance branching successively without significant crossing, with a dendritic field 180–350 µm in diameter In vertical sections, dendrites of ON ganglion cells (ONGCs) stratified near 70% of the IPL depth, and those of the OFF ganglion cells (OFFGCs) stratified near 30% of the IPL depth (Peichl, 1989; Doi et al., 1995).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Light responses of ONGCs are mediated primarily by a cation current of mixed rod and cone inputs
Under infrared visual guidance in dark-adapted flat-mount mouse retinas, we selected ON ganglion cells, with large cell bodies that exhibited increased spike activity during light illumination with loose-patch electrodes. These cells were subsequently recorded with patch electrodes filled with internal solution and Lucifer yellow in the whole-cell voltage-clamp configuration. ONGCs were identified by their three-dimensional morphology revealed by Lucifer yellow fluorescent images in retinal flat mounts and vertical sections (for details, see Materials and Methods). Figure 1, A and B, shows the stacked confocal fluorescent image of an ONGC in the flatmount retina (A) and the image of the same cell in the vertical retinal section (B). The fluorescent images exhibited typical ONGC morphology with dendrites stratifying near 70% of the IPL depth and a dendritic field of 257 µm. The dendrites of all 28 ONGCs we studied stratified near 65–80% of the IPL depth, with field diameters ranging from 210 to 335 µm. Figure 1C shows the light-evoked current responses of the same cell to a 2.5 sec light step recorded under dark-adapted conditions at various holding potentials. ONGCs exhibited two light-evoked current components at the light onset. The early component (the peak inward current, marked with in the inset in D) reversed near +10 mV [close to the equilibrium potential of a cation channel (E_C), as determined by the reversal potential of glutamate-induced current in the presence of 2 mM Co²⁺ (data not shown)] and the late component (the peak outward current, marked with in the inset in D) reversed near -60 mV [the equilibrium potential of the chloride channel (E_Cl); see Materials and Methods]. The current–voltage (I–V) relationships of these two current components (Fig. 1D) have positive slopes, indicating that they are associated with conductance increases. All 28 ONGCs exhibited very similar response waveform, with the reversal potential of the early component ranging from -10 to 20 mV and that of the late component ranging from -46 to -61 mV. The average zero-current potential in darkness of these cells was -63 ± 6 mV (see Materials and Methods). Because ONGCs in the mammalian retina received glutamatergic inputs from DBC_Cs and GABAergic–glycinergic inputs from amacrine cells (Pourcho and Owczarzak, 1989; Cohen et al., 1994; Qin and Pourcho, 1996; Brandstatter et al., 1998), the early component is likely to be mediated by bipolar cell inputs that gate a cation conductance, and the late component is likely mediated by amacrine cell inputs that gate a chloride conductance (Cohen and Miller, 1994).

View larger version (53K): [in this window] [in a new window]   Figure 1. ONGC. A, Stacked confocal fluorescent image in the flat-mount retina; B, image of the same cell in the vertical retinal section. Scale bars, 20 µm. PRL, Photoreceptor layer; OPL, outer plexiform layer; INL, inner nuclear layer; GCL, ganglion cell layer. C, Light-evoked current responses to a 2.5 sec light step (500 nm; -3 = 700 Rh*rod-1sec-1) at various holding potentials. D, Current–voltage relationships of the early () and late () component of the light responses. Spike activities (E), light-evoked excitatory cation current (IC) recorded at ECl (F), and light-evoked inhibitory chloride current (ICl) recorded at EC to 500 nm light steps (2.5 sec) of various intensities (G). H, Response–intensity relationships of the light-evoked cation and chloride currents [IC–Log I () and ICl–Log I ()]. The average dynamic range for IC is 4.9 log units and that for ICl is 5.3 log units.

 

To determine the relative rod–cone contribution to the ganglion cell light responses, we examined the light sensitivity of ONGCs to 500 nm light steps under dark-adapted conditions. We used 500 nm light because mouse rods and cones exhibit the largest sensitivity difference near this wavelength (Lyubarsky et al., 1999), making it easier to distinguish between the two photoreceptor inputs to ganglion cells. Additionally, previous work and our preliminary data from the mouse retina have provided response thresholds and dynamic ranges of rods, rod bipolar cells, some cone bipolar cells, and amacrine cells to 500 nm lights, as well as the relative cone–rod sensitivity (Table 1, top). The threshold of dark-adapted mouse rods for 500 nm light is 1.9 Rh^*rod^-1sec^-1 (Field and Rieke, 2002; Howes et al., 2002), and that of the M-cones is 25–100 times higher (Baylor, 1987; Schneeweis and Schnapf, 1995; Lyubarsky et al., 1999). Because all mouse cones contain the M-pigment and most cones contain the S-pigment of various proportions (Applebury et al., 2000), these cells may be approximately divided into M-dominated (M/s) and S-dominated (S/m) cones, and the S-pigment is 2 log units less sensitive to the 500 nm light than the M-pigment (Lyubarsky et al., 1999). The threshold of rod bipolar cells (DBC_R) in dark-adapted mouse retina is near 0.1–0.6 Rh^*rod^-1sec^-1 (Field and Rieke, 2002), with a dynamic range of 2.2 log units (Berntson and Taylor, 2000). The dynamic range of the M/s cone ON bipolar cell (DBC_MC) response is much wider (4 log units), and it consists of a rod input component with a response threshold near 1 Rh^*rod^-1sec^-1 (presumably mediated by rod–cone coupling and/or DBC_R–AII amacrine cell–DBC_C pathway) and a cone input component with a threshold 2 log units higher (Berntson and Taylor, 2000). We recorded from two HBC_Cs that were more sensitive to 360 nm light than to 500 nm light (data not shown), and thus we believe that they are S/m cone bipolar cells (HBC_SCs). The threshold of these cells for 500 nm light was 2000 Rh^*rod^-1sec^-1, and the dynamic range was 1.5 log units. We also recorded from seven AII amacrine cells (data are not shown but will be presented in a later publication), and the response threshold was 0.001 Rh^*rod^-1sec^-1 and the dynamic range was 3.7 log units.

View this table: [in this window] [in a new window]   Table 1. Response thresholds and dynamic ranges of various mouse retinal neurons

 

By comparing the response thresholds and dynamic ranges of preganglion cells with those of the ganglion cells, we determined what types of bipolar cells and amacrine cells mediate ganglion cell light responses. Figure 1E–H shows the spike activities (E), light-evoked excitatory cation current (I_C) recorded at E_Cl (F), and light-evoked inhibitory chloride current (I_Cl) recorded at E_C (G) of the same ONGC (as in Fig. 1A–D) to 500 nm light steps (2.5 sec) of various intensities. In darkness, the cell exhibited no observable spontaneous spikes. The -7 light step elicited no spike response but a very small I_C, whereas the -6 (0.7 Rh^*rod^-1sec^-1) light step gave rise to a burst of spikes and an inward I_C of 100 pA at E_Cl. As the light step became brighter, the spike train became longer and of higher frequency, and the inward I_C became larger. We measured the light sensitivity of I_C to 500 nm light in all 28 ONGCs, and the average ± SD response–intensity (I_C–Log I) relationsships are plotted in Figure 1H (). The solid curve was fitted by the Hill equation (see Materials and Methods). The average threshold (defined as eliciting 5% of the maximum response) was near -7.2 (0.044 Rh^*rod^-1sec^-1), and the dynamic range was 4.9 log units. Our data demonstrate that the dynamic range and response threshold of I_C in mouse ONGCs are close to those of the DBC_MC (Table 1), which have mixed rod/M-cone signals [two limbs in the response intensity curve (Berntson and Taylor, 2000)], consistent with the idea that the primary light-evoked excitatory inputs in ONGCs are mediated by the cone ON bipolar cells (Bloomfield and Miller, 1986; Bloomfield and Dacheux, 2001).

The average threshold of light-evoked inhibitory current I_Cl of the 28 ONGCs was -6.8 (0.1 Rh^*rod^-1sec^-1), and the average ± SD response–intensity (I_Cl–Log I) relationship (shown in Fig. 1H, ) had an average dynamic range of 5.3 log units. This indicates that the amacrine cells mediating I_Cl of ONGCs have similar rod–cone signals (we named these cells AC_M1) as the DBC_MCs, with a slightly lower threshold and wider dynamic range. In all ONGCs, the threshold of light-evoked spike activities (0.047 ± 0.005 Rh^*rod^-1sec^-1) was closer to that of the I_C than to I_Cl. This is consistent with our observation that the dark resting potential of the ONGCs (-63 ± 6 mV) are very close to E_Cl, and thus the spiking signals of the cells are predominately mediated by I_C from DBC_MC inputs. Average response thresholds and dynamic range as well as the dark membrane potentials of ONGCs are listed in Table 1 (bottom).

Light-evoked chloride currents from amacrine cells shunt ONGC light responses
Despite the homogeneity in the threshold and dynamic range of light-evoked spikes, I_C and I_Cl in all ONGCs, the strength (amplitude) of I_Cl varied from cell to cell. Figure 2A shows histograms of the peak I_Cl amplitudes of 23 dark-adapted ONGCs. The amplitude of I_Cl in approximately one-half of the ONGCs was <50 pA, whereas that of the other one-half was between 60 and 300 pA. There were no noticeable differences in the cell morphology, response sensitivity, I_C amplitude, or I–V relationships between those cells with large I_Cl and those with small I_Cl. However, the frequency of light-evoked spikes in ONGCs with small I_Cl was consistently higher than that with larger I_Cl. Figure 2B shows light-evoked current responses at different potentials in an ONGC with small I_Cl, and Figure 2C shows the spike responses to 500 nm light of different intensities. The I_Cl measured at E_C = + 12 mV was 3 pA, and the spike activities of the cell exhibited the same threshold (-6.3) to 500 nm light as the cell in Figure 1 that had a larger I_Cl; however, the cell displayed a higher spike frequency. Light-elicited spike frequency of the five ONGCs with I_Cl <10 pA was approximately two to five times higher than those ONGCs with larger I_Cl. This inverse relationship between spike frequency and I_Cl suggests that an important function of amacrine cell (AC_M1) inputs (associated with a chloride conductance increase) to ONGCs is to shunt the voltage responses elicited by I_C from bipolar cell inputs. Variations of AC_M1 inputs result in a spectrum of light spike response frequencies in various ONGCs.

View larger version (17K): [in this window] [in a new window]   Figure 2. ICl amplitude and ONGC responses. A, Histogram of ICl amplitude of 23 ONGCs; B, light-evoked current responses of an ONGC (whose ICl peak amplitude was 3 pA) to a 2.5 sec light step (500 nm; -3 = 700 Rh*rod-1sec-1) at various holding potentials; C, the spike responses to 500 nm, 2.5 sec light steps of various intensities of the same cell.

 

Light responses of transient OFFGCs are mediated primarily by a transient cation current from S-cone-driven bipolar cells
By using the same experimental procedures, we studied light responses of 32 OFF ganglion cells. Two major types of OFF response patterns were observed, although the morphology of these cells was very similar. The first type was quiet in darkness (no spontaneous spikes), and they exhibited transient spike activities only at the cessation of the light step [we named these cells transient OFF cells (tOFFGCs)], whereas the second type exhibited spontaneous spikes in darkness and a sustained reduction of spike activity during the light illumination [we named these cells sustained OFF cells (sOFFGCs)]. Among the 32 OFF -like ganglion cells from which we recorded, 12 showed tOFFGC responses and 20 displayed sOFFGC responses.

Figure 3A–D shows the stacked confocal fluorescent image of a tOFFGC in the flat-mount retina (A), the image of the same cell in vertical retinal section (B), the light-evoked current responses to a 2.5 sec 500 nm light step recorded under dark-adapted conditions at various holding potentials (C), and the current–voltage relationships of the responses at light onset and offset (D). The fluorescent images in A and B exhibited typical OFF -cell-like morphology with dendritic field diameter ranging from 180 to 250 µm, and dendrites stratified near 30% (instead of 70% in the ONGCs) of the IPL depth (Peichl, 1989; Doi et al., 1995). The baseline currents at -60 mV or below were very smooth, except for a few small transient inward current bumps [spontaneous EPSCs (sEPSCs)] mediated by spontaneous release of glutamatergic vesicles (Tian et al., 1998). The baseline currents at -40 mV or above, however, were much noisier, with many transient outward currents [spontaneous IPSCs (sIPSCs)]. This abrupt voltage-dependent increase of sIPSCs occurred between -40 and -60 mV and was consistent in all OFF ganglion cells (both tOFFGCs and sOFFGCs; see below). At negative holding potentials (-60 to -100 mV), the light step elicited small outward current responses at the onset and a large transient inward current at the offset. The ON response became larger at more positive potentials, and it did not exhibit a reversal potential between -100 and +60 mV, suggesting that it may be mediated by two opponent conductance changes: a cation conductance decrease at the HBC–tOFFGC synapse and a chloride conductance increase at the AC–tOFFGC synapse. The OFF response was a transient inward current at negative potentials, it became smaller as the holding potential became more positive, and it reversed near +20 mV (E_C). All 12 tOFFGCs exhibited very similar baseline noise and ON and OFF light responses with the E_C values for the OFF response varying from -10 to +25 mV. The average zero-current potential in darkness of these cells was -61 ± 7 mV. These results suggest that the OFF response of the tOFFGC is probably mediated by a cation conductance (with reversal potential near 0 mV), a mechanism that is consistent with anatomical results, suggesting that OFF ganglion cells in mammalian retinas receive synaptic inputs from the OFF cone bipolar cells (HBC_Cs) (Bloomfield and Dacheux, 2001).

View larger version (57K): [in this window] [in a new window]   Figure 3. tOFFGC. A, Stacked confocal fluorescent image in the flat-mount retina; B, image of the same cell in the vertical retinal section. Scale bars, 20 µm. PRL, Photoreceptor layer; OPL, outer plexiform layer; INL, inner nuclear layer; GCL, ganglion cell layer. C, Light-evoked current responses to a 2.5 sec light step (500 nm; -2 = 7000 Rh*rod-1sec-1) at various holding potentials; D, current–voltage relationships of the ON () and OFF () responses. Spike activities (E), light-evoked excitatory cation current (IC) recorded at ECl (F), and light-evoked inhibitory chloride current (ICl) recorded at EC to 500 nm light steps (2.5 sec) of various intensities (G). H, Response–intensity relationships of the light-evoked cation and chloride currents [ON IC–Log I (), OFF IC–Log I (), and ON ICl–Log I ()]. The average dynamic range for ON IC is 3.9 log units, that for the OFF IC is 1.6 log units, and that for ICl is 3.3 log units.

 

Figure 3E–G shows the spike activities (E), I_C (F), and I_Cl recorded at E_C (G) of a tOFFGC to 500 nm light steps (2.5 sec) of various intensities. In darkness, the cell exhibited no observable spontaneous spikes but some sEPSCs. The -5 step elicited no spiking activities and a very small ON I_C, whereas the -4 and -3 light steps evoked no spikes and larger outward ON I_C. Brighter light steps (-2 and -1) elicited a brief train of spikes and a transient OFF inward I_C, in addition to the outward ON I_C. We measured the light sensitivity of OFF I_C to 500 nm light in all tOFFGCs, and the average ± SD response–intensity (OFF I_C–Log I) relationships are plotted in Figure 3H (). The solid curve was fitted by the Hill equation. The average threshold was near -2.7 (1394 Rh^*rod^-1sec^-1), and the dynamic range was 1.6 log units. The average spike response threshold was 1813 ± 470 Rh^*rod^-1sec^-1, indicating that the spike response of these cells are mediated by the OFF I_C. Because the OFF I_C and spike response threshold and dynamic range are very close to those of the HBC_SCs (Table 1), we believe that they are primarily mediated by the S/m cones through the S/m cone hyperpolarizing bipolar cells.

"Silent" bipolar cell inputs to tOFFGCs at light onset
The threshold of the ON I_C (Fig. 3F,H, ) was approximately -5, which was 2–3 log units higher than that of the OFF I_C and spike responses. We measured the light sensitivity of ON I_C to 500 nm light in all 12 tOFFGCs, and the average ± SD response–intensity (ON I_C–Log I) relationship is plotted (and fitted by the Hill equation) in Figure 3H (). The average threshold was near -5.8 (1.1 Rh^*rod^-1sec^-1), and the average dynamic range was 3.9 log units. By comparing these results with the parameters listed in Table 1, we believe that the ON I_C of tOFFGCs is mediated primarily by HBC_MCs. It is important to note that ON I_C is an outward current that results in membrane hyperpolarization and decrease of spike activity. Therefore, because tOFFGCs do not exhibit spontaneous spike activities in darkness (Fig. 3E), ON I_C cannot decrease spiking further, and thus it has no physiological consequences on the tOFFGC output and can be considered as a silent bipolar cell input.

Silent amacrine cell inputs to tOFFGCs
The average threshold of I_Cl elicited by 500 nm light from 12 tOFFGCs was -5.5 (2.2 Rh^*rod^-1sec^-1), and the average ± SD response–intensity (I_Cl–Log I) relationships (Fig. 3H) had a dynamic range of 3.3. Comparing these results with the parameters listed in Table 1 reveals that the I_Cl of tOFFGCs is probably mediated by amacrine cells with mixed rod and cone inputs (we named these cells AC_M2, which receive inputs from M-cone bipolar cells with mixed rod) [possibly through rod–cone coupling and/or AII amacrine cells (Demb and Pugh, 2002)] and M-cone inputs. Because the average zero-current potential in darkness of these cells was -61 ± 7 mV, a value very close to E_Cl, the AC_M2-mediated current response (I_Cl) contributes little to the total light-evoked current. Moreover, because tOFFGCs do not exhibit spontaneous spike activities in darkness (Fig. 3E) and I_Cl returns to the baseline before the onset of the transient inward OFF I_C (Fig. 3F,G; for shorter light stimuli, OFF I_C was much smaller), the shunting action of I_Cl-mediated conductance increase causes little physiological consequence to the tOFFGC output and can be considered as a silent amacrine cell input.

Average response thresholds and dynamic range as well as the dark membrane potentials of ON I_C, OFF I_C, and I_Cl of the tOFFGCs are listed in Table 1 (bottom).

Light responses of sOFFGCs are mediated by both a cone-driven bipolar cell input and a rod-driven amacrine cell input
Figure 4A–C shows the stacked confocal fluorescent image of a sustained sOFFGC in the flat-mount retina (A), the image of the same cell in vertical retinal section (B), and the light-evoked current responses to a 500 nm 2.5 sec light step recorded under dark-adapted conditions at various holding potentials (C). The fluorescent images in A and B exhibited typical OFF ganglion cell morphology with dendrites stratified near 30% of the IPL depth (Peichl, 1989; Doi et al., 1995). Similar to the tOFFGCs, the baseline currents at -60 mV or below were relatively smooth and at -40 mV or above were much noisier, possibly attributable to electrical coupling with amacrine cells that release GABAergic or glycinergic vesicles onto the recorded cell (Xin and Bloomfield, 1997) (see Discussion). The light step gave rise to a transient outward current followed by a sustained outward current at all potentials (without a reversal potential), and the current–voltage relationships of the peak and steady-state light responses are shown in Figure 4D. The lack of reversal potential suggests that the light response is probably mediated by two opponent conductance changes, a cation conductance decrease at the HBC–sOFFGC synapse and a chloride conductance increase at the AC–sOFFGC synapse. The sustained outward current lasted for seconds after light offset and then exhibited one to two transient dips (Fig. 4C). All 20 sOFFGCs exhibited very similar baseline noise and light response patterns, and the average zero-current potential in darkness of these cells was -51 ± 7 mV. A major difference between the tOFFGCs and sOFFGC is that sOFFGCs did not show the transient inward current at light offset (compare Figs. 3C, 4C).

View larger version (59K): [in this window] [in a new window]   Figure 4. sOFFGC. A, Stacked confocal fluorescent image in the flat-mount retina; B, image of the same cell in the vertical retinal section. Scale bars, 20 µm. PRL, Photoreceptor layer; OPL, outer plexiform layer; INL, inner nuclear layer; GCL, ganglion cell layer. C, Light-evoked current responses to a 2.5 sec light step (500 nm; -2 = 7000 Rh*rod-1sec-1) at various holding potentials; D, current–voltage relationships of the peak () and steady-state component () of the light responses. Spike activities (E), light-evoked excitatory cation current (IC) recorded at ECl (F), and light-evoked inhibitory chloride current (ICl) recorded at EC to 500 nm light steps (2.5 sec) of various intensities (G). H, Response–intensity relationships of the light-evoked peak cation and chloride currents [IC–Log I () and ICl–Log I (). The average dynamic range for IC is 3.0 log units, and that for ICl is 5.9 log units.

 

Figure 4 E–H shows the spike activities (E), I_C (F), and I_Cl (G) of the same sOFFGC as in Figure 4 A–D to 500 nm light steps (2.5 sec) of various intensities. In darkness, the cell exhibited spontaneous spikes of 5–10 Hz and some sEPSCs at E_Cl. The -8 step resulted in a very brief period of decrease in spiking activities, elicited no I_C, but evoked a sustained outward I_Cl. The -7 light step decreased the spike frequency for 0.7 sec, elicited no I_C, and evoked a larger I_Cl. Brighter light steps resulted in longer periods of spike decrease and larger I_Cl, and, at -5, light started to elicit I_C. As the light step became even brighter, the decrease of spike activity lasted longer, and I_C and I_Cl became larger and longer. We measured the light sensitivity of I_C and I_Cl to 500 nm light in all 20 sOFFGCs, and the average ± SD response–intensity (I_C–Log I and I_Cl–Log I) relationships are plotted and fitted with the Hill equation in Figure 4H. The average threshold for I_C was near -6.1 (0.55 Rh^*rod^-1sec^-1) with a dynamic range of 3.0 log units, and the average threshold for I_Cl was near -8.5 (0.0022 Rh^*rod^-1sec^-1) with a dynamic range of 5.9 log units. The average threshold of the spike decrease was -8.0 (0.007 Rh^*rod^-1sec^-1). These results suggest that I_C of sOFFGCs is probably mediated by HBC_MCs, similar to the bipolar cells that mediate I_C in tOFFGCs. I_Cl of sOFFGCs is likely to be mediated by the AII amacrine cells who have very high sensitivity to 500 nm lights (Table 1). Because the dynamic range of I_Cl is much wider than that of the AII ACs, another AC with mixed rod and cone inputs (possibly AC_M2) may also be involved in mediating I_Cl in sOFFGCs. Because the threshold of spike response was closer to I_Cl than to I_C, I_Cl should contribute significantly more to the spike responses. This may be partially explained by our observation that the average dark membrane potential of sOFFGCs was near -51 mV, 10 mV more depolarized than E_Cl. This is in contrast to the ONGCs and tOFFGCs whose average dark membrane potentials are much closer to E_Cl, and thus, in these cells, I_Cl could only contribute to the light response by voltage shunting. The difference in dark membrane potential may also explain why sOFFGCs exhibit spontaneous spikes in darkness whereas ONGCs and tOFFGCs do not.

Average response thresholds and dynamic range as well as the dark membrane potentials of ON I_C and I_Cl of the sOFFGCs are listed in Table 1 (bottom).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mouse ganglion cells exhibit three distinct types of light responses: ON, transient OFF, and sustained OFF
By using loose-patch, whole-cell voltage-clamp and Lucifer yellow fluorescent techniques, we studied the light responses of 51 ganglion cells with -cell-like morphology in dark-adapted mouse retina and found that they can be clearly divided into three types. The first type, the ONGCs, exhibits no spike activity in darkness, increased spikes in light, sustained light-evoked inward cation current (I_C) at E_Cl, sustained light-evoked chloride current (I_Cl) of varying amplitude at E_C, and large soma (20–25 µm in diameter) with -cell-like dendritic field 180–350 µm stratifying near 70% of the IPL depth. The second type, the tOFFGCs, exhibit no spike activity in darkness, transient increased spikes at light offset, small sustained light-evoked outward I_C in light and large transient inward I_C at light offset, and a sustained outward I_Cl in light. Morphologically, the tOFFGCs are similar to the ONGCs except for that their dendrites stratified near 30% of the IPL depth. The third type, the sOFFGCs, exhibit maintained spike activity of 5–10 Hz in darkness, sustained decrease spikes in light, sustained outward I_C, sustained outward I_Cl in light, and a morphology similar to the tOFFGCs.

Our data agree with the ON and OFF sublaminar rule of the retinal IPL very well: ganglion cells with dendrites ramified in sublamina A display OFF responses and those with dendrites in sublaminar B display ON responses (Nelson et al., 1978). On the other hand, ganglion cells in mammalian retinas are believed to have transient light responses (Cleland et al., 1975; Peichl and Wassle, 1981; Saito, 1983); however, in the mouse retina, we found that only one-quarter of the ganglion cells are transient (tOFFGCs). This difference may result from species difference or different adaptational conditions (all of our recordings were performed under infrared illumination). Alternatively, because the transient light responses described in previous studies are correlated with ganglion cells of large somas without detailed dendritic morphology, they may represent other populations of ganglion cells with large somas.

Synaptic circuitries of GCs: each type of GCs receive light-evoked signals from BCs and ACs with different rod/cone inputs
In this study, we used the whole-cell voltage clamp technique to separate the light-evoked BC and AC inputs (I_C and I_Cl, respectively) to GCs. By comparing the response threshold and dynamic ranges of I_C and I_Cl and spike activities of each type of GCs with the corresponding parameters of preganglion cells, we proposed a functional synaptic circuitry of the BC and AC inputs to GCs (Fig. 5). It is worth noting that this circuitry diagram includes a minimum number of circuit components, although our data may also be explained by more complex schemes. The outlines of our proposed circuitry are consistent with the general plan set forth by anatomical analysis but with several new and more detailed findings revealing how synapses in the inner retina function.

View larger version (36K): [in this window] [in a new window]   Figure 5. Synaptic circuit diagram of the ONGCs, tOFFGCs, and sOFFGCs in the mouse retina. R, Rods; C(M/s), M-pigment-dominated cones; C(S/m), S-pigment-dominated cones; DBCR, rod depolarizing bipolar cell; DBCMC, M-cone-dominated depolarizing bipolar cell; HBCMC, M-cone-dominated hyperpolarizing bipolar cell; HBCSC, S-cone-dominated bipolar cells; AII, AII amacrine cells; ACM1, amacrine cell with mixed rod–cone inputs in the ON GC pathway; ACM2, amacrine cell with mixed rod–cone inputs in the OFFGC pathway; arrows, chemical synapses (red, glutamatergic; blue, GABAergic/glycinergic; + sign, preserving; and - sign, inverting); (red), electrical synapses; PRL, photoreceptor layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer (a, sublamina a; b, sublamina b); GCL, ganglion cell layer.

 

We found that the light responses of ONGCs are quite homogenous, and they all appear to receive excitatory inputs from DBC_MCs, which give a mixed rod/M-cone signal with a rod-like threshold and a wide (combined rod–cone) dynamic range. The rod signals are mediated by either the rod–DBC_R–AII–DBC_C (ON1) pathway or the rod–cone–DBC_C (ON2) pathway (Bloomfield and Dacheux, 2001; Demb and Pugh, 2002), and the cone signals are mediated directly by the M-cone–DBC_MC synapse. In all 28 ONGCs, we never observed a response threshold higher then 20 Rh^*rod^-1sec^-1 or an operating range beyond 1000 Rh^*rod^-1sec^-1, suggesting that the contribution of the S-cone inputs is minor. The only significant variation among ONGCs is their spike response frequency and the amplitude (not the threshold or dynamic range) of I_Cl. Because the dark membrane potential of these ganglion cells is close to E_Cl, we suggest that the primary function of the amacrine cell (AC_M1) input is to shunt the DBC_MC-mediated input. The inverse relationship between I_Cl amplitude and the frequency of light-evoked spikes in the ONGCs supports this notion.

Our finding that all ONGCs exhibit homogenous response–intensity function (Fig. 1H) contrasts a recent study that reports four groups of ON ganglion cells in the mouse retina, each having a distinct response–intensity curve (Deans et al., 2002). Because responses of these ganglion cells were recorded with extracellular electrodes and the cell morphology was not revealed, the four groups may reflect both and non- ON ganglion cells (and perhaps spiking displaced amacrine cells). On the basis of the response threshold and dynamic range, it appears that ONGCs belong to the groups with intermediate sensitivity and wide operating range (mixed rod–cone inputs) and not those of high (rod driven) or low sensitivity (cone driven).

The two types of OFFGCs display drastically different responses, and thus their BC and AC inputs are different. tOFFGCs exhibit transient increase of spikes at light offset with a very high threshold, and the sOFFGCs exhibit spike decrease when the light is turned on with an extremely low threshold. These cells seem to receive two excitatory bipolar cell inputs: one is mediated by a sustained HBC with an M-cone-dominated signal and the other by a transient HBC (with an off overshoot response) with an S-cone-dominated signal (J.-J. Pang and S. M. Wu, unpublished data) and an AC input with mixed rod/M-cone inputs (AC_M2). The HBC_SC-mediated transient OFF I_C is responsible for the transient OFF spike response. The HBC_MC-mediated ON I_C and AC-mediated I_Cl are both inhibitory (because they are outward currents), and, because tOFFGCs do not exhibit spontaneous spikes in darkness, these outward currents do not serve much physiological function (silent synapses). On the other hand, sOFFGC responses are extremely sensitive to 500 nm light, with a threshold lower than HBC_MCs and even lower than HBC_Rs (Field and Rieke, 2002). It appears that sOFFGC responses are mediated primarily by the AII AC-mediated I_Cl, which has a threshold of near 0.001 Rh^*rod^-1sec^-1 (Pang and Wu, unpublished data) at the low light intensity range. An HBC_MC-mediated I_C is also involved in mediating the spike responses of the cells at higher light intensities. These results also suggest that the feedforward synapse from AII AC to sOFFGCs are stronger (or of higher gain) than the feedback synapse from AII AC to HBC_MCs and the electrical synapse between AII AC and DBC_MCs (Fig. 5), because the AII response to dim light (below 0.01 Rh^*rod^-1sec^-1) could only be observed in I_Cl of the sOFFGCs.

A major difference between sOFFGCs and the other two types of GCs is that sOFFGCs exhibit spontaneous spike activity of 5–10 Hz in darkness, whereas the other GCs do not. This can be partially explained by our finding that sOFFGCs have an average dark membrane potential (-51 ± 7 mV) 10 mV more positive than the other two types of GCs (-63 ± 6 and -61 ± 7 mV for the ONGCs and tOFFGCs, respectively). Another factor that may contribute to the spontaneous spiking in sOFFGCs is that it has been shown, at least in the salamander retina, that the sustained OFF bipolar cells exhibit large sEPSCs in darkness (Wu et al., 2000). These sEPSCs in HBCs may trigger sEPSCs in sOFFGCs. As sOFFGCs are maintained at a relatively more depolarized voltage in darkness, large sEPSCs may depolarize the cell above the threshold of action potentials and thus cause spontaneous spike activities.

In our voltage-clamp experiments, we found an abrupt voltage-dependent increase of sIPSCs that occurred between -60 and -40 mV in all OFFGCs (both tOFFGCs and sOFFGCs) but not in ONGCs. One possible explanation for this abrupt voltage-dependent increase of sIPSCs is that the depolarizing current needed to maintain the positive holding potential may leak into amacrine cells through gap junctions (Xin and Bloomfield, 1997), which would facilitate the release of GABAergic or glycinergic vesicles from amacrine cells to the recorded ganglion cell (Tian et al., 1998). Anatomical studies have shown that reciprocal electrical synapses exist between OFFGCs and GABAergic ACs in mammalian retinas (Dacey and Brace, 1992; Jacoby et al., 1996; Bloomfield and Xin, 1997). Our voltage-clamp results suggest that membrane depolarization in OFFGCs, such as occurs during action potentials, may cause considerable depolarizing current flow into ACs (AC_M2) through the gap junctions. Because AC neurotransmitters are inhibitory, this reciprocal electrical synapse may serve as a negative feedback circuit for spiking activities in the OFFGCs (Fig. 5).

In summary, the synaptic circuitry diagram (Fig. 5) derived from our study is primarily consistent with the general plan for ON and OFF ganglion cells set forth by anatomical studies, and thus our results provide the first physiological support for the organization found in the mouse retina. Additionally, our data elucidate which synapses serve physiological function and which ones do not, as well as which synapses are more dominant than others in mediating the light responses of the cells. For example, our data suggest that the primary function of the AC inputs to ONGCs is to regulate the spiking frequency of the cells by voltage shunting and that the AII AC feedforward synapse is the dominant inputs for sOFFGCs, at least within the low light intensity range. In addition, the outward ON I_C and ON I_Cl from HBC_MC and ACs to tOFFGCs serve little physiological function, and the transient inward OFF I_C from HBC_SCs is the dominant input for the tOFFGCs spike response. Finally, OFFGCs, rather than ONGCs, are likely to make functional reciprocal electrical synapses with ACs. These physiological characteristics of GCs could not be revealed by the previous anatomical studies, and thus they provide new insights on our understanding of the functional circuitry of the mammalian retina.

	Footnotes

 
Received Mar. 17, 2003; revised Apr. 29, 2003; accepted May. 1, 2003.

This work was supported by National Institutes of Health Grant EY 04446, National Institutes of Health Vision Core Grant EY 02520, the Retina Research Foundation (Houston, TX), and Research to Prevent Blindness. We thank Drs. Laura Frishman, Helga Kolb, Susan Cushman, and Roy Jacoby for critically reading this manuscript.

Correspondence should be addressed to Dr. Samuel M. Wu, Cullen Eye Institute, Baylor College of Medicine, One Baylor Plaza, NC-205, Houston, TX 77030. E-mail: swu{at}bcm.tmc.edu.

Copyright © 2003 Society for Neuroscience 0270-6474/03/236063-11$15.00/0

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