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. 2002 Feb 15;539(Pt 1):239–251. doi: 10.1113/jphysiol.2001.013110

Non-linear, high-gain and sustained-to-transient signal transmission from rods to amacrine cells in dark-adapted retina of Ambystoma

Xiong-Li Yang 1, Fan Gao 1, Samuel M Wu 1
PMCID: PMC2290137  PMID: 11850516

Abstract

In darkness, On-Off amacrine cells (ACs) of the tiger salamander retina exhibited large spontaneous transient depolarizing potentials (sTDPs) with average peak amplitude of 5.05 ± 2.5 mV and average frequency of 0.42 ± 0.25 s−1. Under voltage-clamp conditions the cell displayed large spontaneous postsynaptic currents (sPSCs) with average peak amplitude of 98 ± 39 pA and average frequency of 0.45 ± 0.22 s−1. To a light step, ACs gave rise to a transient ‘On’ response at the light onset and a transient ‘Off’ response at light offset, followed by a train of TDPs (‘After’ response). Near the response threshold (0.3 activated rhodopsin molecules per rod per second), light-evoked TDPs (leTDPs) of similar amplitude and kinetics as the large sTDPs observed in darkness were seen, and about half of these leTDPs elicited a regenerative potential (RP). Brighter light steps gave rise to more leTDPs and higher rates of RPs in the On, Off and After responses. Within the linear response range of the rods, the AC response was non-linear, with the highest gain (676 ± 429) near the dark potential. The amplitude of Off responses increased with the duration of the light step, and ACs may use this to encode speeds of moving stimuli: the faster the light object moves, the smaller the AC Off response. Moreover, the number of leTDPs in the AC After response increased with light intensity, and the onset of the After response coincides with bipolar cell tail response recovery. One possible origin of the large sTDPs and leTDPs is the spontaneous and depolarization-induced regenerative calcium potentials (RCaPs) in bipolar cell synaptic terminals. RCaPs in bipolar cell synaptic terminals cause transient glutamate release that results in the sTDPs in darkness, and leTDPs in On, Off and After responses in ACs.

The visual system is an extremely sensitive light detector. Only a few photons are needed to produce a conscious visual sensation (Hecht et al. 1942). This high sensitivity is partially mediated by non-linear, high-gain synaptic pathways such as the synapses between outer retinal neurons and inner retinal neurons (Copenhagen et al. 1987, 1990). Under dark-adapted conditions, voltage responses of neurons in the outer retina to dim lights are linearly related to the light intensity, and the average voltage gains of the rod-second-order cell synapses range from 5 to 20 (Baylor & Hodgkin, 1973; Yang & Wu, 1996, 1997). The response-intensity functions of ganglion cells in the inner retina within this light intensity range are non-linear, with average rod-mediated voltage gains over 100 (Baylor & Fettiplace, 1977; Copenhagen et al. 1990). The sensitivity and voltage gains of the rod-amacrine cell synaptic pathway, on the other hand, have not been systematically studied. Since amacrine cells (ACs) are the interneurons in the inner retina that make synapses onto bipolar cell axon terminals, ganglion cells and other ACs (Wong-Riley, 1974), they may play crucial roles in mediating non-linear activities in the inner retina. In this study, we investigate the spontaneous transient voltage and current events in darkness and light-evoked responses of ACs with intracellular voltage recording and whole-cell voltage-clamp techniques. Response-intensity relationships, step sensitivity, and voltage gains are systematically studied. Based on the similarities of the spontaneous transient events in darkness and in light-evoked responses, we propose a model that may qualitatively account for the non-linear and high-gain signal transfer from outer retina to inner retina.

To a maintained step of light, neurons in the outer retina give rise to sustained voltage responses, whereas the majority of neurons in the inner retina display transient depolarizing responses at light onset and offset (Werblin & Dowling, 1969; Kaneko, 1970). Sustained light responses encode stationary visual images, and transient responses register moving objects in the visual world (Dowling, 1987). Several mechanisms have been suggested to carry out the sustained-to-transient signal transformation (Miller, 1979; Maguire et al. 1989; Lukasiewicz et al. 1995). While many factors may contribute to shaping the transient light responses in ACs and ganglion cells, results described here provide an additional mechanism for the sustained-to-transient signal transformation. The transient On and Off responses of ACs may be mediated by bursts of transient depolarizing potentials (TDPs). Synaptic events underlying the TDPs and their roles in mediating the non-linear characteristics of AC responses are described.

METHODS

Preparations

Larval tiger salamanders (Ambystoma tigrinum) purchased from Charles E. Sullivan Co. (Nashville, TN, USA) and KONS Scientific Co. Inc. (Germantown, WI, USA) were used in this study in accordance with the National Institute of Health and Baylor College of Medicine Animal Care guidelines. The procedures of dissection and making retinal slices have been described previously (Werblin, 1978; Wu, 1987). Before each experiment, salamanders were anaesthetized in MS-222 until the animals gave no visible response to touch or water vibration. The animals were then quickly decapitated and the eyes enucleated. Flat-mounted whole retinas were inverted over a hole in the Millipore filter paper so that microscope light could pass through. Dissection and tissue preparation were done under infrared illumination with a dual-unit Fine-R-Scope (FJW Industry, Mount Prospect, IL, USA). Oxygenated Ringer solution was introduced continuously to the superfusion chamber, and the control Ringer solution contained 108 mmNaCl, 2.5 mm KCl, 1.2 mm MgCl2, 2 mm CaCl2 and 5 mm Hepes (adjusted to pH 7.7). All chemicals were dissolved in control Ringer solution. The retina (photoreceptor side up) or retinal slices were viewed with a Zeiss ×40 water immersion lens or a ×32 objective lens modified for Hoffman modulation contrast optics (Hoffman Modulation Optics, Greenvale, NY, USA). During the experiment, retinal cells and the electrode were clearly observed, and in the light response experiments a TV monitor connected to the infrared image converter (model 4415; COHU, Palo Alto, CA, USA) attached to the microscope was used.

Recording

Intracellular recordings were made with micropipettes drawn with a modified Livingston puller with Omega Dot tubing (1.0 mm o.d. and 0.5 mm i.d.). The micropipettes were filled with 2 m potassium acetate and had tip resistances measured in Ringer solution of 100–600 MΩ. Voltage-clamp recordings were made with an Axopatch 200A amplifier connected to a DigiData 1200 interface, and data acquisition and analysis were processed by pCLAMP 6.1 software (Axon Instruments, Foster City, CA, USA). Amplifier signals were low-pass filtered at 2 kHz with a 4-pole Bessel filter and sampled at 10 kHz. Patch electrodes of 5 MΩ tip resistance when filled with internal solution containing 118 mm caesium methanesulfonate, 10 mm CsCl, 5 mm EGTA, 0.5 mm CaCl2, 1 mm MgCl2, 4 mm ATP, 0.3 mm GTP, 10 mm Tris and 0.8 mm Lucifer Yellow, adjusted to pH 7.2 with CsOH were made with Narishige or Kopf patch electrode pullers. The input resistances of the cells were usually 100–500 MΩ and the series resistances were typically 10 MΩ (uncompensated, which would lead to a clamp voltage error of 1–2 mV for the largest responses). The chloride equilibrium potential, ECl, with this internal solution is about −60 mV. Estimates of the liquid junction potential at the tip of the patch electrode prior to seal formation varied from −9.2 to −9.6 mV. For simplicity, we have corrected all holding potentials in this paper by 10 mV. Amacrine cells were identified by their morphology when filled with Lucifer Yellow, and their characteristic light responses (Wu & Maple, 1998).

Light source

The flat-mounted isolated retina or retinal slice was stimulated with a dual-beam photostimulator. Two independent light beams, whose intensity and wavelength could be adjusted by neutral-density filters and interference filters, were provided by quartz halogen sources. The light was transmitted to the preparation by way of the epi-illuminator and the objective lens of the microscope, and the spot diameter on the retina could be adjusted by a diaphragm in the epi-illuminator. In most experiments described, whole-field illumination (1000–1500 μm in diameter) was used. Measurements of the step sensitivity of ACs and voltage gain of the rod-AC pathway were carried out in the flat-mounted isolated retina preparation with photoreceptor side up, thus incident light beams were parallel to the axes of the photoreceptor outer segments. A typical tiger salamander rod outer segment is 25 μm long and 10 μm in diameter (Diamond & Copenhagen, 1995). Taking a specific axial density of rhodopsin of 0.015 μm−1 and quantum efficiency for photoisomerization of 0.67 (Dartnall, 1968; Liebman & Entine, 1968), the photoisomerization cross-section (PIC) can be calculated by the following equation (Yang & Wu, 1996):

graphic file with name tjp0539-0239-mu1.jpg

Thus 1 photon μm−2 s−1 is approximately equivalent to 30 Rh* (activated rhodopsin molecules) rod−1 s−1. The intensity of light sources was measured with a radiometric detector (United Detector Technology, Santa Monica, CA, USA). The intensity of unattenuated 500 nm light (log I = 0) was 2.05 × 107 photons μm−2 s−1.

Since the axes of photoreceptors in retinal slices are perpendicular to the direction of incident light, the PIC calculation for the flat-mounted retina does not hold in slices. We calibrated the intensity of light stimulus for the slice preparation by adjusting the unattenuated light so that a −7.3, 500 nm, 0.5 s light step elicited a 1 mV rod response, which is the average rod response in flat-mounted retinas to a 500 nm, 0.5 s light step, with −7.3 log units attenuation of the 2.05 × 107 photons μm−2 s−1 light beam (Yang & Wu, 1996).

RESULTS

Light responses of dark-adapted On-Off amacrine cells

Over 85 % of the amacrine cells (ACs) we recorded from the dark-adapted salamander retina were On-Off cells that exhibited a transient depolarizing response at the light onset (On response) followed by a slow decay that returned to the cell's dark membrane potential in about 7–10 s (in cases when light steps were long enough), and a transient depolarizing response at the light offset (Off response) followed by a train of discrete transient depolarizing potentials (TDPs) (After response). The amplitude of the Off response and the number of TDPs in the After response varied with the duration of light stimuli. In darkness, the resting membrane potential of ACs ranged from −70 to −87 mV. Figure 1A shows an example of such On-Off AC responses to light steps (500 nm, −7.3) of various durations. In response to each light step, the cell exhibited a transient On response (○) with peak amplitude near 19 mV, which decayed to the baseline in about 9 s. The Off response (•) to the long light steps (7–10 s) was about 15–17 mV. For shorter light steps, which were turned off before the On response decayed to the dark level, the cell exhibited a smaller Off response whose amplitude was roughly proportional to the level of the On response decay. The Off responses were followed by trains of discrete TDPs (After responses, ▾). We repeated the same experiment in eleven other ACs and the average On and Off response amplitudes to light steps of various durations are plotted in Fig. 1B. To a 0.5 s light step, the average Off response was only about 20 % of the On response, and the Off response reached a steady-state level (about 80 % of the On response) as the light duration became 5 s or longer.

Figure 1. Dependence of amacrine cell light responses on light duration.

Figure 1

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A, voltage responses of an On-Off amacrine cell recorded from a dark-adapted isolated retina to light steps (500 nm, −7.3) of various durations. In response to each light step, the cell exhibited a transient On response (○), a transient Off response (•) followed by an After response (trains of TDPs, ▾). B, average On and Off response amplitudes of 12 On-Off amacrine cells vs. the duration of light steps. Error bars indicate standard deviations.

Response-intensity relationships, step sensitivity and voltage gains of the rod-mediated amacrine cell responses

We next studied the amacrine cell voltage responses to light steps of various intensities. In order to study rod-mediated inputs to ACs, we examined responses of rods, rod-dominated bipolar cells and ACs to dim light steps. Figure 2 shows the voltage responses of a rod, a rod-dominated depolarizing bipolar cell (DBCR), a rod-dominated hyperpolarizing bipolar cell (HBCR), and an On-Off AC recorded under dark-adapted conditions to 500 nm light steps (-9.3 to −5.3 log units attenuation, 0.5 s in duration). Cones were unresponsive to these dim lights (Yang & Wu, 1996) and thus the DBCR, HBCR and AC responses were completely mediated by rods. The rod, DBCR and HBCR responses were sustained during the light step, whereas the AC responded with transient depolarizations at light onset and offset. Dark-adapted ACs were extremely sensitive to the 500 nm light step, with a threshold near 9.3 log units of attenuation. The amplitude of the On response increased with light intensity and reached its maximum level near −8.3 to −7.3. Most cells maintained the maximum amplitude but a few cells exhibited amplitude decrease at brighter light intensities. The response-intensity (V-log I) relationships of the On responses of thirteen dark-adapted ACs are shown in Fig. 3A. In addition to its dependence on light duration (see Fig. 1), the Off response also varied with light intensity: its amplitude first increased, then decreased, and in some cells increased again as the light intensity became brighter. The V-log I relationships of the Off responses of the thirteen ACs are shown in Fig. 3B. The AC After responses also varied with the light intensity. The number of discrete TDPs and the duration of the train increased with the light intensity. Additionally, there was a quiescent gap (where no TDPs occurred, horizontal lines between two arrows in Fig. 2) between the Off response and the After response, and the duration of these gaps increased with the light intensity and occurred during the DBCR and HBCR response tails (see Fig. 2).

Figure 2. Rod, bipolar cell and amacrine cell light responses.

Figure 2

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Voltage responses of a rod, a rod-dominated depolarizing bipolar cell (DBCR), a rod-dominated hyperpolarizing bipolar cell (HBCR), and an On-Off amacrine cell recorded in the dark-adapted isolated retina to dim whole-field 500 nm light steps (-9.3 to −5.3 log units attenuation and 0.5 s in duration). The amacrine cell displayed a transient On response (○) with a delay that became shorter as the light became brighter, a transient Off response (•) and an After response. The number of discrete TDPs and the duration of the train in the After response increased with the light intensity. There was a quiescent gap (where no TDPs occurred, bars between two arrows) between the Off response and the After response, and the duration of these gaps increased with the light intensity and overlapped with the duration of the DBCR and HBCR response tails.

Figure 3. Response-intensity (V-log I) relationships of the On (A) and Off (B) responses of dark-adapted amacrine cells.

Figure 3

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The On response amplitude of the majority of the 13 cells increased with the light intensity (except 3 cells whose amplitude first increased and then decreased with increasing light intensity). The Off response of almost all amacrine cells varied with light intensity: its amplitude first increased, then decreased, and in some cells increased again as the light intensity became brighter.

Previous studies have shown that voltage responses of the rod-dominated bipolar cell under dark-adapted conditions vary widely from cell to cell, with rod-mediated voltage gains ranging from 1.5 to 50 (obtained by the ratio of the bipolar cell responses to the rod responses to 500 nm, −8.3 light) (Yang & Wu, 1997). Since we do not know the relative contribution of various types of bipolar cells to the On-Off ACs, it is difficult to determine bipolar AC voltage gains by the ratio of their light responses. On the other hand, the rod responses in dark-adapted tiger salamander retina are quite homogenous, with response variations less than 30 % (Yang & Wu, 1997). Therefore, we characterized the voltage gain of the rod-AC pathway. Table 1 gives the response amplitudes of fifteen On-Off ACs recorded under dark-adapted conditions to 500 nm, −9.3, −8.3 and −7.3 light steps. From an earlier study, the rod response in dark-adapted salamander retina to the 500 nm −7.3 light step is 1.04 ± 0.31 mV, and that to the −8.3 light step is 0.11 ± 0.06 mV (Yang & Wu, 1996). Since rod responses less than 1 mV lay within the linear voltage-intensity range (in which the response amplitude is proportional to the light intensity) (Baylor & Hodgkin, 1973), the rod response to a 500 nm, −9.3 light step can be estimated as 0.01 mV. The step sensitivity, SS, defined as ΔV/IS (ΔV is the peak voltage response elicited by a light step of intensity IS), of rods within this linear range is constant (about 0.03 mV Rh*−1 s rod (Yang & Wu, 1996)). Since the Off and After responses of ACs exhibit complex patterns of intensity- and duration-dependence (Figs 1–4), we estimated the step sensitivity and voltage gain of the rod-AC pathway by comparing the rod response only with the peak AC On response. SS of these ACs (mean ± s.d.) was 21 ± 13.8 mV Rh*−1 s rod (range 6.45–47.1) for the −9.3 light step, 5.39 ± 1.7 mV Rh*−1 s rod (range 1.74–7.25) for the −8.3 light step, and 0.55 ± 0.15 mV Rh*−1 s rod (range 0.25–0.74) for the −7.3 light step (see Table 1). This progressive decline of the SS values with light intensity is caused by AC response saturation at very dim light levels, and it contrasts with the constant SS of the rods, horizontal cells and bipolar cells within this intensity range (Yang & Wu, 1996, 1997). Consequently, signal transmission from the outer retina to the inner retina within the rod linear response range is highly non-linear. The rod-AC voltage gain for light onset, GOn, defined as ΔVAC(On)/ΔVrod for each light step, is also listed in Table 1. For the −9.3 light step, GOn (mean ± s.d.) was 676 ± 429 (range 200–1450), for the −8.3 light step, GOn was 152 ± 48 (range 49–205), and for the −7.3 light step, GOn was 16.5 ± 4.5 (range 7–22).

Table 1.

Amacrine cell light responses and voltage gains

500nm,−9.3,Is =0.31 Rh* s−1 rod−1ΔVrod =0.01 mV 500nm,−8.3,Is =3.1 Rh* s−1 rod−1ΔVrod =0.11 mV 500nm,−7.3,Is =31 Rh* s−1 rod−1ΔVrod =1.04 mV
AC No. ΔVAC(On)(mV) GOn ΔVAC(Off)(mV) ΔVAC(On)(mV) GOn ΔVAC(Off)(mV) ΔVAC(On)(mV) GOn ΔVAC(Off)(mV)
1 12.5 1250 10.0 20.5 186 18.7 21.3 20 18.9
2 3.8 380 5.0 17.2 156 15.8 17.1 16 16.0
3 13.3 1330 13.8 21.5 195 20.8 22.1 21 21.4
4 14.6 1460 4.6 19.5 177 18.3 20.0 19 21.4
5 4.0 400 9.0 16.7 152 10.0 18.0 17 9.0
6 2.0 200 12.9 14.8 135 15.7 15.7 15 3.3
7 6.3 630 8.4 21.9 199 21.9 22.5 22 8.8
8 2.0 200 5.0 15.0 136 4.7 14.0 13 5.0
9 11.0 1100 10.2 22.0 200 20.0 22.0 21 9.0
10 2.5 250 2.0 7.5 68 7.5 8.5 8 11.0
11 6.0 600 6.0 22.5 205 23.0 23.0 22 16.6
12 10.0 1000 9.5 20.9 190 17.5 19.2 18 14.0
13 4.1 410 5.6 10.0 91 10.0 14.8 14 13.5
14 2.0 200 6.2 5.4 49 6.3 7.6 7 6.3
15 7.3 730 8.0 15.0 136 14.0 16.0 15 10.0
Mean 6.76 676 16.72 152 17.16 16.5
s.d. 4.29 429 5.28 48 4.68 4.5
ΔSOn (ΔVOn/IS)(mean ±s.d.)(range) 21 ± 13.8mV Rh*−1s rod (6.45−47.1 mV Rh*−1s rod) 5.39 ± 1.7 mV Rh*−1s rod (1.74−7.25 mV Rh*−1s rod) 0.55 ± 0.15 mVRh*−1s rod (0.25 −0.74 mV Rh*−1s rod)
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ΔVrod: light-evoked voltage response of the rod;Is:intensity of the light step;ΔVAC(On): amacrine cell voltage response to light onset; ΔVAC(Off):amacrine cell voltage response to light offset;GOn:voltage gain of the rod–AC synapse for light onset; ΔSOn: step sensitivity of amacrine cells for light onset.

Figure 4. Spontaneous and light-evoked TDPs.

Figure 4

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A, discrete spontaneous transient depolarizing potentials (sTDPs) of an amacrine cell recorded from a dark-adapted isolated retina. Large sTDPs whose peak amplitude was greater than 2.5 mV are marked by arrows, and the sTDP with a spike on the top is marked by *. B, voltage responses of the same amacrine cell to 9 repetitive stimuli of a very dim (500 nm, 1 s, −9.3, about 0.3 Rh* rod−1 s−1) light step. A transient depolarizing potential occurred about 0.75 s after the light onset (On response, ○) and another occurred about 1 s after the light offset (Off response, •). C, voltage responses of the same amacrine cell to brighter light steps (−8.3, −7.3, −6.3 and−-5.3); the cell exhibited a transient On response (○), an Off response (•) of variable size, and an After response (▾). D, second response in the second row in B (-9.3, marked 1) and the first response in C (-8.3, marked 2) on a faster time scale. RPs are marked with *.

Spontaneous transient depolarizing potentials (sTDPs) in darkness and light-evoked transient depolarizing potentials (leTDPs)

In addition to light-evoked TDPs (leTDPs) in On, Off and After responses, ACs exhibited spontaneous TDPs (sTDPs) in darkness. Figure 4A shows the sTDPs of an AC in darkness. Complete analysis of size distribution, kinetics and frequency of sTDPs is beyond the scope of this paper. In this study, we only considered the large sTDPs whose peak amplitude was greater than 2.5 mV (arrows in Fig. 4A), because they were easier to recognize, and as we will show next, the amplitude and time course of these large sTDPs are similar to the leTDPs. These large sTDPs varied in size (2.5–8 mV), and they occurred randomly, occasionally in bursts, and a few had regenerative potentials (RPs) present on the top (*). The average (± s.d.) frequency of these large sTDPs was 0.42 ± 0.25 s−1, and the mean amplitude of these large events was 5.05 ± 2.5 mV (total large sTDP events = 368 in five ACs). Figure 4B shows the voltage responses to nine repetitive stimuli of a very dim light step (500 nm, 1 s, −9.3, about 0.3 Rh* rod−1 s−1, see Methods). A leTDP occurred about 0.75 s after the light onset (On response, ○) and another occurred about 1 s after the light offset (Off response, •). The amplitudes and kinetics of these leTDPs are similar to the large sTDPs (arrows in Fig. 4A), but a much higher percentage (about 50 %) elicited a RP. Light stimuli dimmer than −9.3 (e.g. −9.67) never evoked any responses (there was no correlation between the light stimuli and the spontaneously occurring large sTDPs, and the records were indistinguishable from the records obtained in darkness). Therefore the −9.3, 500 nm light step was the threshold light stimulus of these ACs. It is important to note that all nine light steps in Fig. 4B never elicited responses whose amplitude was substantially smaller than the large sTDPs in darkness, and thus these large sTDPs seem to be the smallest unit (or elementary event) that constitutes the light responses. It appears that each of the nine light steps triggered a leTDP (or a burst of leTDPs) at its onset (0.75 s after) and offset (1 s after) by activating the same synaptic mechanisms that generate the large sTDPs. In response to brighter light steps (Fig. 4C), the cell exhibited a transient On response (○) of larger amplitude (it triggered more leTDPs that almost always elicited RPs) and shorter time delays, an Off response (•) of variable size, and an After response (▾) containing leTDPs of similar amplitude and time course as the large sTDPs. The number of leTDPs in the After response increased with the intensity of the light stimuli.

In order to closely examine the RPs on top of the leTDPs, we show in Fig. 4D the voltage responses to a −9.3 light step (Fig. 4B, second row, middle response) and the −8.3 light step (Fig. 4C, first row, first response) on a faster time scale. The RPs (marked by *) on top of the leTDPs had rise times of 30–50 ms and decay times of 50–100 ms, which are one to two orders of magnitude slower than the time course of a typical sodium action potential (Hodgkin & Huxley, 1952; Diamond & Copenhagen, 1995). These RPs were not affected by bath application of 1–2 μm tetrodotoxin (TTX) (not shown), and thus they are unlikely to be mediated by the TTX-sensitive sodium action potentials. In about 10 % of the On-Off ACs recorded from dark-adapted salamander retinas, however, TTX-sensitive action potentials of faster time courses (2–5 ms in duration) on top of the On and Off RPs were observed. These action potentials were often labile and they only lasted for several minutes after cell impalement (Vallerga, 1981).

Voltage-clamp analysis of spontaneous and light-evoked transient postsynaptic currents

Results in the previous section suggest that AC light responses (leTDPs) may be mediated by the same synaptic mechanisms underlying the large sTDPs in darkness. We therefore characterized the large sTDPs in darkness and the large leTDPs by the whole-cell voltage-clamp technique. Figure 5A shows the voltage-clamp currents in darkness at various holding potentials from an On-Off AC in the salamander retinal slice. At all holding potentials, discrete spontaneous postsynaptic currents (sPSCs) that resembled the waveform and frequency of the large sTDPs (arrows in Fig. 4A) were observed. At potentials near or below −60 mV, all sPSCs were inward; between −60 and 0 mV, some sPSCs were inward, whereas others were outward; near or above 0 mV, all sPSCs were outward. This suggests that there may be two types of sPSCs, the excitatory sPSCs (sEPSCs) with a reversal potential near 0 mV, and the inhibitory sPSCs (sIPSCs) with a reversal potential near −60 mV (ECl, see Methods). Since ACs in the salamander retina receive glutamatergic excitatory synaptic inputs from bipolar cells and GABAergic and glycinergic inhibitory inputs from other ACs (Yang & Yazulla, 1988a, b; Dixon & Copenhagen, 1992; Wu & Maple, 1998), we used antagonists of glutamate, GABA and glycine receptors to separate the two types of sPSCs. Figure 5B shows that when 100 μm picrotoxin and 1 μm strychnine were applied to the AC, the frequency of the sPSCs was reduced, and all sPSCs reversed near 0 mV, consistent with the idea that when GABAergic and glycinergic sIPSCs were blocked, only sEPSCs were present. The addition of 10 μm 6,7-dinitro-quinoxaline-2,3-dione (DNQX) and 50 μm d-aminophosphonovalerate (AP5) completely blocked these sEPSCs (Fig. 5C). Picrotoxin or strychnine alone did not block all sIPSCs, and DNQX or AP5 alone did not block all sEPSCs (not shown), indicating that both GABAergic and glycinergic synapses are involved in mediating the sIPSCs, and both AMPA/kainate and NMDA receptors are involved in mediating the sEPSCs. The sIPSCs and sEPSCs recovered after the cell was washed with normal Ringer solution for 5 min (Fig. 5D). We repeated these experiments in six other On-Off ACs and all cells gave similar results.

Figure 5. Effects of GABA and glycine receptor antagonists on spontaneous PSCs.

Figure 5

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A, voltage-clamp currents in darkness at various holding potentials from an amacrine cell in the salamander retinal slice. At all holding potentials, discrete spontaneous postsynaptic currents (sPSCs) that resembled the waveform and frequency of the spontaneous TDPs (Fig. 4A) were observed. B, effects of 100 μm picrotoxin (PTX) + 1 μm strychnine (STR) on the same cell at the same set of holding potentials. PTX + STR blocked all sIPSCs. C, effects of 10 μm DNQX + 50 μm AP5 on the same cell at the same set of holding potentials. DNQX + AP5 blocked sEPSCs. Both sEPSCs and sIPSCs recovered after wash with normal Ringer solution (D).

At holding potentials near the AC dark potential (-70 to −80 mV), the average (± s.d.) frequency of the large sPSCs (peak amplitude > 50 pA) was 0.45 ± 0.22 s−1, and the mean amplitude of these large events was 98 ± 39 pA (total large sPSC events = 725 in seven ACs). The average frequency of these large sPSCs agrees very well with the average large sTDP frequency (0.42 ± 0.25 s−1) described above. The input impedance of a salamander AC in slices at negative potentials recorded with microelectrodes was about 50 MΩ (ranging from 10 to 100 MΩ, S. M. Wu, unpublished results), and thus an average large sPSC gives a voltage change of 4.9 mV (98 pA × 50 MΩ), which is very close to the average amplitude of the large sTDP (5.05 ± 2.5 mV) recorded from dark-adapted ACs with microelectrodes (see above). It is worth noting that the large sPSCs never exhibited a regenerative component as in some of the large sTDPs (* in Fig. 4A), suggesting that RPs on top of the large sTDPs are mediated by voltage-dependent mechanisms in ACs that cannot be activated under voltage-clamp conditions.

Figure 6 shows the voltage-clamp current responses of an AC to a 0.5 s light step (500 nm) of various intensities at a holding potential of −80 mV. In darkness, the cell exhibited sPSCs (top trace), and to the −9.3 light step (second trace) it responded with a transient PSC about 0.45 s after the light onset and a second transient PSC 0.55 s after the light offset. These light-evoked PSCs were of similar amplitude as the large sPSCs in darkness. This is consistent with the idea that the light step triggered a large PSC (or a burst of large PSCs) at its onset and offset by the same mechanisms that triggered large sPSCs. As the light became brighter, the On responses became larger (they triggered more large PSCs) and had shorter delays, the Off response first became larger and then smaller, and the number of transient large PSCs in the After response became larger. Additionally, there was a quiescent gap (where no PSCs occurred, horizontal line between two arrows) between the Off response and the After response. These features are very similar to the On, Off and After voltage responses of ACs to light stimuli shown in Fig. 2 and Fig. 3. It is important to note again that there was no regenerative component in the light-evoked current responses to light steps of any intensity (whereas the voltage responses to light brighter than −8.3 always exhibited RPs, see Figs 1, 2 and 4), indicating that the RPs present on top of the light-evoked TDPs are mediated by voltage-dependent mechanisms in ACs that are not activated under voltage-clamp conditions.

Figure 6. Light-evoked PSCs in an amacrine cell.

Figure 6

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Voltage-clamp current of an amacrine cell in a retinal slice in darkness (top trace) and current responses to a 0.5 s light step (500 nm) of various intensities at a holding potential of−80 mV. In darkness, the cell exhibited spontaneous PSCs, and it responded to the −9.3 light step with a transient PSC about 0.9 s after the light onset and a second transient PSC 1.05 s after the light offset. These light-evoked PSCs were of similar amplitude and time course as the sPSCs in darkness. As the light became brighter, the On responses (○) became larger with shorter delays, the Off response (•) first became larger and then smaller, and the number of transient PSCs in the After response (▾) became larger. There was a quiescent gap (horizontal line between two arrows) between the Off response and the After response.

The voltage dependence of the current responses to a 0.5 s light step (500 nm, −7.3) from a dark-adapted salamander AC is shown in Fig. 7A. At all holding potentials, the cell exhibited a transient On response and a train of transient PSCs in the After response, but not much Off response (due to the intensity and duration of the light step used, see Figs 1, 2, 4 and 6). At potentials near or below −60 mV, current responses were inward; between −60 and 0 mV, the On responses were biphasic (inward and outward) and some transient PSCs in the After response were inward, whereas others were outward; near or above 0 mV, all current responses were outward. This pattern of voltage dependence of light-evoked responses is exactly the same as the pattern of the sPSCs in darkness shown in Fig. 5, suggesting that the light-evoked responses and the spontaneous large PSCs in darkness are mediated by similar synaptic mechanisms. In order to test this assertion, we used the same glutamate, GABA and glycine receptor antagonists as in Fig. 5 to study the AC light responses. Figure 7B shows that when 100 μm picrotoxin and 1 μm strychnine was applied to the AC in Fig. 7A, the On response reversed near 0 mV and the response kinetics became much slower. The number of transient PSCs in the After response was reduced, and they reversed near 0 mV. This is consistent with the idea that when GABAergic and glycinergic inputs were blocked, light responses were mediated by the excitatory synapses. The addition of 10 μm DNQX and 50 μm AP5 almost completely blocked the On responses and the transient PSCs in the After response (Fig. 7C). The On responses and the transient PSCs recovered after the cell was washed with normal Ringer solution for 5 min (Fig. 7D). We repeated these experiments in eight other ACs, and they all showed similar responses to picrotoxin, strychnine, DNQX and AP5.

Figure 7. Effects of GABA and glycine receptor antagonists on light-evoked PSCs.

Figure 7

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A, voltage-clamp current responses to a 0.5 s light step (500 nm, −7.3) at various holding potentials of a amacrine cell in a dark-adapted retinal slice. B, effects of 100 μm picrotoxin (PTX) + 1 μm strychnine (STR) on the same cell at the same set of holding potentials. In the presence of PTX + STR, the On response kinetics became much slower, the number of transient PSCs in the After response was reduced, and both the On responses and PSCs reversed near 0 mV. C, effects of 10 μm DNQX + 50 μm AP5 on the same cell at the same set of holding potentials. DNQX + AP5 blocked the On responses and the PSCs in the After response. The On responses and the transient PSCs recovered after the cell was washed with normal Ringer solution for 5 min (D).

Amplitude and kinetics of individual transient PSCs in darkness, in the On, Off and After responses

In order to quantitatively compare the large PSCs in darkness and in light responses, we measured the peak amplitude (A), time-to-peak (τ0) and decay time constant (τD) of 725 discrete large PSCs in darkness, 59 discrete large PSCs in On responses, 78 discrete large PSCs in Off responses, and 289 large discrete PSCs in After responses at a holding potential of −80 mV. Examples of large PSCs are shown in Fig. 8A. We selected discrete large PSCs exhibiting a fast monophasic rise and a slower decay that could be fitted by a single exponential function (exponential fits of the decay time constants are shown as thick lines in Fig. 8B). Large PSCs with multiple peaks were considered as asynchronous multiples of single events, and were not analysed. The average (± s.d.) peak amplitudes (A, in pA at −80 mV) are 98 ± 39, 109 ± 45, 88 ± 25 and 95 ± 34 for the PSCs in darkness, and in On, Off and After responses, respectively, the times-to-peak (τ0, in ms) are 4.2 ± 1.1, 4.2 ± 1.3, 3.6 ± 1.1 and 3.7 ± 1.0, and the decay time constants (τD, the time constant of the single exponential decay, in ms) are 8.8 ± 2.1, 7.1 ± 1.8, 8.9 ± 3.0 and 9.4 ± 1.7. These results indicate that the average amplitude and kinetics of the large PSCs in darkness, in On, Off and After responses of amacrine cells are very similar, suggesting that these large PSCs may be mediated by the same synaptic mechanisms.

Figure 8. Kinetics of PSCs.

Figure 8

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A, examples of large PSCs in darkness, in On, Off and After responses. The decay time constants (τD) of each PSC fitted by a single exponential (thick line) are shown in B.

DISCUSSION

The rod-amacrine cell synaptic pathway is non-linear with high voltage gain

We have shown in this study that light-evoked responses of On-Off ACs in dark-adapted salamander retina exhibit several non-linear characteristics. In the rod linear response- intensity range where horizontal cells and bipolar cells exhibit linear responses (Yang & Wu, 1996, 1997), the AC On response is highly non-linear with higher voltage gain for dimmer lights. The average voltage gain of the rod-AC pathway near the dark rod potential is 676, which is about forty times higher than the average voltage gain of the rod-rod bipolar cell synapses (Yang & Wu, 1997). This rod-AC voltage gain is of the same order of magnitude as the rod-ganglion cell voltage gain (Copenhagen et al. 1990), suggesting that similar mechanisms are involved in mediating the light responses of both ACs and ganglion cells.

The absolute threshold of the visual system is defined as the light stimulus that can produce 55 % ‘frequency-of-seeing’ (Davson, 1990). Our results show that to repetitive stimuli of the −9.3, 500 nm (0.3 Rh* rod−1 s−1) light step, about half of the AC responses are large leTDPs without RPs, whose amplitudes are similar to those of the sTDPs, and thus they may not be distinguishable from spontaneous voltage fluctuations in darkness. The other half of the AC responses to the −9.3 light step are leTDPs with RPs, whose amplitude is substantially larger than the sTDPs in darkness, and thus they may register light. To the onset of brighter light steps, leTDPs are always with RPs (Fig. 2 and Fig. 4). Therefore the −9.3, 500 nm light is close to the absolute threshold of dark-adapted ACs for registering light-evoked signals. Since the voltage gain of the rod- ganglion cell synaptic pathway is of the same order of magnitude as the voltage gain of the rod-AC pathway (Copenhagen et al. 1990), the absolute threshold of dark-adapted salamander ganglion cells may also be close to 0.3 Rh* rod−1 s−1. It is of great interest to compare the absolute thresholds of neurons in the inner retina with the absolute threshold of the salamander visual system, and behavioural studies are needed to unravel the latter.

Since ACs exert inhibitory actions on ganglion cells and bipolar cell axon terminals (Werblin & Dowling, 1969; Gao et al. 2000), the large AC responses near visual threshold may reduce the light responses of ganglion cells and bipolar cells. This appears to counter the high gain signalling in the rod-ganglion cell pathway (Baylor & Fettiplace, 1977; Copenhagen et al. 1990). However, most ganglion cells and HBCs in salamander retina exhibit spontaneous postsynaptic currents (sPSCs) (Gao & Wu, 1999; Gao et al. 2000; Wu et al. 2000); the large AC inputs may serve to reduce the spontaneous voltage fluctuations in ganglion cells and bipolar cells and hence to improve the signal-to-noise ratio in these cells near visual threshold. Additionally, because the temporal and spatial profiles of the AC and bipolar cell inputs to ganglion cells are different (Werblin, 1972), the inhibitory AC inputs may not always counter the excitatory bipolar cell inputs in all ganglion cells.

Time-dependent non-linearities of amacrine cell Off and After responses

In addition to the non-linear high voltage gain, ACs exhibit time-dependent non-linearities in their Off and After responses. Off responses to short light steps are small, become increasingly larger (with higher chance of generating RPs) as the duration of the light step increases, and reach steady state when the light step is near 5 s (Fig. 1B). It is possible that ACs use this duration-dependent Off response to encode speeds of moving stimuli. For example, if the time for a light bar sweeping across the receptive field of an AC is 0.5 s, the Off response will be small, but if the time for the same light bar to move across the AC receptive field is 5 s, then the Off response will be large (see Fig. 1). Therefore the On-Off ACs may serve as speed detectors for moving stimuli: within the dynamic range of the duration-Off response relationship (0–5 s, Fig. 1B), the speed of a moving stimulus is registered as the amplitude of the AC's Off response; the faster the light moves, the smaller the AC Off response. Further work is needed to determine how ganglion cells and higher-order visual neurons process the AC Off signals, and how duration- and speed-dependent information is registered in the visual system.

Another time-dependent process in On-Off ACs is that the duration of the After response increases with light intensity (Fig. 2). Since the onset of the AC After response coincides with bipolar cell tail recovery, AC After responses are most likely to be mediated by the HBCs. This is because the output synapses of both DBCs and HBCs are sign-preserving (Werblin & Dowling, 1969; Miller, 1979), and thus the AC After responses should be triggered by the depolarizing ramp of the HBC tail recovery, not by the hyperpolarizing ramp of the DBC tail recovery. After-images have been observed in human subjects after the cessation of flashes in darkness, and the duration of afterimages increases with light intensity (Macleod & Hayhoe, 1974). It is important to determine whether After responses similar to those in ACs are present in ganglion cells, and how they are related to the afterimages experienced by humans.

Possible roles of the transient depolarizing potentials (TDPs) in mediating the non-linear characteristics of the rod-AC pathway

Our data show that large TDPs (and large PSCs) in darkness, in On, Off and After responses in On-Off ACs are of similar amplitude and kinetics, and thus they may be mediated by the same synaptic mechanism. One possible origin of these large TDPs is the spontaneous and depolarization-induced regenerative calcium potentials (RCaPs) observed in bipolar cell synaptic terminals (but not in the cell bodies) (Burrone & Lagnado, 1997; Zenisek & Matthews, 1998; Protti et al. 2000). Although RCaPs have not been recorded from salamander bipolar cell axon terminals (as they are much smaller than those of the goldfish, and like the goldfish, RCaPs are not present in bipolar cell bodies (Wu et al. 2000)), it is reasonable to postulate that they may exist there. Spontaneous postsynaptic currents in darkness (Gao et al. 2000; Wu et al. 2000) as well as the depolarizing phases of the bipolar cell light responses (onset of the DBC, offset of the HBC, and the recovery of the voltage tail of the HBC) trigger RCaPs in bipolar cell synaptic terminals (Zenisek & Matthews, 1998). These RCaPs induce transient bursts of glutamate release from bipolar cell axon terminals (von Gersdorff et al. 1998) that result in the sTDPs (in darkness), and leTDPs in On, Off and After responses in ACs.

Since RCaPs are all-or-none (Burrone & Lagnado, 1997; Zenisek & Matthews, 1998), the large TDPs are not graded. The −9.3 light step only polarizes the rod by about 0.01 mV and the rod bipolar cells by about 0.16 mV (both are extrapolated values), but it triggers RCaPs in bipolar cell synaptic terminals, which generate large leTDPs in ACs. Large leTDPs without RPs have average amplitudes of about 5 mV and the leTDPs with RPs have average amplitudes of 12–20 mV (Fig. 4). This may explain the unusual high voltage gain of the rod-AC synaptic pathway. The bipolar AC signals are amplified twice by non-linear boosters: the RCaPs in the bipolar cell synaptic terminals and the RPs in ACs. As light becomes brighter (−8.3 and −7.3), rod and rod bipolar cell responses increase linearly (Yang & Wu, 1996, 1997), and more RCaPs are triggered in bipolar cell synaptic terminals, which generate more leTDPs at light onset and offset with larger RPs in ACs. Since AC On responses saturate about 1 log unit above the threshold (Fig. 2 and Fig. 4C), the voltage gain decreases sharply for the −8.3 and −7.3 light steps, and thus the AC response-intensity relationship within the rod linear response range is non-linear.

Our voltage clamp results in Fig. 5 and Fig. 7 show that synaptic inputs from other ACs also mediate sTDPs and light-evoked TDPs if the dark membrane potential of the AC is substantially more negative than the chloride equilibrium potential (ECl). Since the resting membrane potential of dark-adapted ACs lay between −70 and −80 mV, and ECl values in ACs are in the range −50 to −70 mV (Miller & Dacheux, 1983), it is likely that at least in some ACs, AC-AC synapses contribute to the sTDPs in darkness and the light-evoked responses, although such AC contribution may be much weaker than the bipolar cell contribution because signals from the latter cells have a larger driving force.

Ganglion cells share the same bipolar cell output synapses with ACs (Wong-Riley, 1974) and possess regenerative action potentials (Diamond & Copenhagen, 1995), so it is likely that their bipolar cell inputs may pass through similar amplification mechanisms. It is important to note, however, that the postsynaptic RPs present on top of the TDPs in On-Off ACs are TTX insensitive, and they exhibit much slower time courses than the sodium action potentials in ganglion cells (Cook & McReynolds, 1998). Ionic mechanisms underlying the AC RPs are not clear, and further investigations are needed to clarify this issue.

The time-dependent non-linearities in the Off and After responses are also consistent with the RCaP scheme. The onset of light step triggers a burst of RCaPs in bipolar cell synaptic terminals, which result in a transient wave of TDPs and RPs in ACs (On response). It takes about 7–9 s for the On response to return to the baseline (Fig. 1), and during this recovery period, the threshold of the RPs in ACs is elevated. Consequently, the Off response during the recovery period is smaller because the probability of RP occurrence is lower. This is consistent with the existence of a refractory period for the RCaPs in bipolar cell synaptic terminals (Protti et al. 2000). The depolarizing ramps of the HBCR at light offset and during the response tail recovery trigger RCaPs (and thus leTDPs and RPs) because membrane hyperpolarization may lower the threshold of the RCaPs. This is reasonable because studies on dissociated goldfish bipolar cells have demonstrated that RCaPs occurring in bipolar cell synaptic terminals can be activated by membrane depolarization with a threshold near −43 mV (Burrone & Lagnado, 1997), and bursts of RCaPs are observed at the cessation of membrane hyperpolarizations (Zenisek & Matthews, 1998). The observation that the duration of the After response increases with light intensity and that the onset of the After response coincides with bipolar cell response tail recovery (Fig. 2) is consistent with this scheme. Depolarization during the HBCR response tail recovery triggers RCaPs in HBCR synaptic terminals (and thus large TDPs and RPs in ACs) since the hyperpolarizing response tails lower the threshold of the RCaPs (von Gersdorff & Matthews, 1999). It is worth noting that although the RCaP scheme discussed above provides a reasonable explanation for the non-linearities of the Off and After responses, other mechanisms, such as dendritic spikes in the amacrine cell network and inhibitory feedback loops in the inner plexiform layer (IPL) (Miller, 1979) may also contribute to the generation of TDPs in amacrine cells.

Sustained-to-transient signal transmission

Our results suggest that the transient On and Off responses in ACs are mediated by transient bursts of large TDPs. Previous studies have suggested three mechanisms for the sustained-to-transient signal transformation in the inner retina. (1) Transient depolarizing responses in ACs result from linear synaptic summation of depolarizing and hyperpolarizing bipolar cell (DBC and HBC) responses, with the sustained components cancelling each other (Miller, 1979). (2) Sustained bipolar cell inputs to ACs and ganglion cells are truncated by a delayed inhibitory input from ACs (Maguire et al. 1989; Nirenberg & Meister, 1997; Dong & Werblin, 1998). (3) Glutamate receptor desensitization makes sustained responses more transient by speeding up the response decay time (Lukasiewicz et al. 1995). Mechanism (1) may be involved in forming the On and Off transient responses in ACs by cancelling the sustained components of the DBC and HBC inputs, but it cannot explain the non-linear high gain and the time-dependent non-linearities of the AC light responses. Mechanisms (2) and (3) may make the AC responses more transient, but application of GABA or glycine antagonists or glutamate receptor desensitization blockers never convert transient responses into sustained signals (Lukasiewicz et al. 1995; Gao et al. 2000; F. Gao & S. M. Wu, unpublished results). Therefore it is possible that all three mechanisms may contribute to the sustained-to-transient signal transformation, but they are unlikely to be the predominant factors. The On and Off responses of ACs are mainly mediated by the large leTDPs induced by transient bursts of RCaPs in bipolar cell synaptic terminals that trigger the large leTDPs and RPs in ACs. Further experiments are needed to verify this assertion.

Acknowledgments

This work was supported by NIH EY 04446, NIH Vision Core EY 02520, the Retina Research Foundation (Houston), and Research to Prevent Blindness, Inc.

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