Much of what is currently known about the visual response of retinal bipolar cells is based on studies of rod-dominant responses to flashes in the dark in the isolated retina. This minireview summarizes quantitative findings on contrast processing in the intact light-adapted retina based on intracellular recording from more than 400 cone-driven bipolar cells in the tiger salamander: 1) In the main, the contrast responses of ON and OFF cells are surprisingly similar, suggesting a need to refine the view that ON and OFF cells provide the selective substrates for processing of positive and negative contrasts, respectively. 2) Overall, the response is quite nonlinear, showing very high gain for small contrasts, some 10–15 times greater than that of cones, but then quickly approaches saturation for higher contrasts. 3) Under optimal conditions of light adaptation, both classes of bipolar cells show evidence for efficient coding with respect to the contrasts in natural images. 4) There is a marked diversity within both the ON and OFF bipolar cell populations and an absence of discrete subtypes. The dynamic ranges bracket the range of contrasts in nature. 5) For both ON and OFF cells, the receptive field organization shows a striking symmetry between center and surround for responses of the same polarity and thus opposite contrast polarities. 6) The latency difference between ON and OFF cells is about 30 ms, which seems qualitatively consistent with a delay due to the G-protein cascade in ON bipolar cells. 7) In sum, we report quantitative evidence for at least 11 transformations in signal processing that occur between the voltage response of cones and the voltage response of bipolar cells.

The role of intracellular organelles in Ca2+ homeostasis was studied in salamander rod and cone photoreceptors under conditions that simulate photoreceptor activation by darkness and light. Sustained depolarization evoked a Ca2+ gradient between the cell body and ellipsoid regions of the inner segment (IS). The standing pattern of calcium fluxes was created by interactions between the plasma membrane, endoplasmic reticulum (ER), and mitochondria. Pharmacological experiments suggested that mitochondria modulate both baseline [Ca2+]i in hyperpolarized cells as well as kinetics of Ca2+ entry via L type Ca2+ channels in cell bodies and ellipsoids of depolarized rods and cones. Inhibition of mitochondrial Ca2+ sequestration by antimycin/oligomycin caused a three-fold reduction in the amount of Ca2+ accumulated into intracellular organelles in both cell bodies and ellipsoids. A further 50% decrease in intracellular Ca2+ content within cell bodies, but not ellipsoids, was observed after suppression of SERCA-mediated Ca2+ uptake into the ER. Inhibition of Ca2+ sequestration into the endoplasmic reticulum by thapsigargin or cyclopiazonic acid decreased the magnitude and kinetics of depolarization-evoked Ca2+ signals in cell bodies of rods and cones and decreased the amount of Ca2+ accumulated into internal stores. These results suggest that steady-state [Ca2+]i in photoreceptors is regulated in a region-specific manner, with the ER contribution predominant in the cell body and mitochondrial buffering [Ca2+] the ellipsoid. Local [Ca2+]i levels are set by interactions between the plasma membrane Ca2+ channels and transporters, ER and mitochondria. Mitochondria are likely to play an essential role in temporal and spatial buffering of photoreceptor Ca2+.

Action potentials in amacrine cells are important for lateral propagation of signals across the inner retina, but it is unclear how many subclasses of amacrine cells contain voltage-gated sodium channels or can fire action potentials. This study investigated the ability of amacrine cells with narrow (< 200 μm) and wide (> 200 μm) dendritic fields to fire action potentials in response to depolarizing current injections and light stimulation. The pattern of action potentials evoked by current injections revealed two distinct classes of amacrine cells; those that responded with a single action potential (single-spiking cells) and those that responded with repetitive action potentials (repetitive-spiking cells). Repetitive-spiking cells differed from single-spiking cells in several regards: Repetitive-spiking cells were more often wide field cells, while single-spiking cells were more often narrow field cells. Repetitive-spiking cells had larger action potential amplitudes, larger peak voltage-gated NaV currents lower action potential thresholds, and needed less current to induce action potentials. However, there was no difference in the input resistance, holding current or time constant of these two classes of cells. The intrinsic capacity to fire action potentials was mirrored in responses to light stimulation; single-spiking amacrine cells infrequently fired action potentials to light steps, while repetitive-spiking amacrine cells frequently fired numerous action potentials. These results indicate that there are two physiologically distinct classes of amacrine cells based on the intrinsic capacity to fire action potentials.

Contrast encoding for sinusoidal modulations of luminance contrast was investigated by intracellular recording in the intact salamander retina. In what appears to be the first study of this kind for vertebrate bipolar cells, responses of the central receptive-field mechanism of cone-driven cells to modulation of 3 Hz were analyzed quantitatively via both signal averaging and a Fast Fourier Transform (FFT) while the retina was light adapted to 20 cd/m2. Depolarizing and hyperpolarizing bipolar cells showed very similar encoding. Both responded with sinusoidal waveforms whose amplitude varied linearly with modulation depths ranging up to 7–8%. The slope of the modulation/response curve was very steep in this range. Thus, the contrast gain was high, reaching values of 6–7, and the half-maximal response was achieved at modulations of 9% or less. At modulations above ∼15%, the responses typically showed strong compressive nonlinearity and the waveform was increasingly distorted. At maximum modulation, the higher harmonics of the FFT constituted about 30% of the amplitude of the fundamental. Measurements were also made for cones and horizontal cells. Both cell types showed predominantly linear responses and low contrast gain, in marked contrast to bipolar cells. These results suggest that the high contrast gain and strong nonlinearity of bipolar cells largely arise postsynaptic to cone transmitter release. Further experiments were performed to compare responses to contrast steps versus those to sinusoidal modulation. In the linear range, we show that the contrast gains of cones and horizontal cells are low and virtually identical for both steps and sinusoidal modulations. In bipolar cells, on the other hand, the contrast gain is about two times greater for steps than that for the 3-Hz sine waves. These results suggest that mechanisms intrinsic to bipolar cells act like a high-pass filter with a short time constant to selectively emphasize contrast transients over slower changes in contrast.

We found that L-glutamate (L-Glu) inhibits L-type Ca2+ currents (ICa) in rod photoreceptors. This inhibition was studied in isolated rods or rods in retinal slices from tiger salamander using perforated patch whole cell recordings and Cl−-imaging techniques. Application of L-Glu inhibited ICa by ∼20% at 0.1 mM and ∼35% at 1 mM. L-Glu also produced an inward current that reversed around ECl. The metabotropic glutamate receptor (mGluR) agonists t-ADA (Group I), DCG-IV (Group II), and L-AP4 (Group III) had no effect on ICa. However, the glutamate transport inhibitor, TBOA (0.1 mM), prevented L-Glu from inhibiting ICa. D-aspartate (D-Asp), a glutamate transporter substrate, also inhibited ICa with significantly more inhibition at 1 mM than 0.1 mM. Using Cl− imaging, L-Glu (0.1–1 mM) and D-Asp (0.1–1 mM) were found to stimulate a Cl− efflux from terminals of isolated rods whereas the ionotropic glutamate receptor agonists NMDA, AMPA, and kainate and the mGluR agonist, 1S,3R-ACPD, did not. Glutamate-evoked Cl− effluxes were blocked by the glutamate transport inhibitors TBOA and DHKA. Cl− efflux inhibits Ca2+ channel activity in rod terminals (Thoreson et al. (2000), Visual Neuroscience17, 197). Consistent with the possibility that glutamate-evoked Cl− efflux may play a role in the inhibition, reducing intraterminal Cl− prevented L-Glu from inhibiting ICa. In summary, the results indicate that activation of glutamate transporters inhibits ICa in rods possibly as a consequence of Cl− efflux. The neurotransmitter L-Glu released from rod terminals might thus provide a negative feedback signal to inhibit further L-Glu release.

To investigate the influence of voltage-sensitive conductances in shaping light-evoked responses of retinal bipolar cells, whole-cell recordings were made in the slice preparation of the tiger salamander, Ambystoma tigrinum. To study contrast encoding, the retina was stimulated with 0.5-s steps of negative and positive contrasts of variable magnitude. In the main, responses recorded under voltage- and current-clamp modes were remarkably similar. In general agreement with past results in the intact retina, the contrast/response curves were relatively steep for small contrasts, thus showing high contrast gain; the dynamic range was narrow, and responses tended to saturate at relatively small contrasts. For ON and OFF cells, linear regression analysis showed that the current response accounted for 83–93% of the variance of the voltage response. Analysis of specific parameters of the contrast/response curve showed that contrast gain was marginally higher for voltage than current in three of four cases, while no significant differences were found for half-maximal contrast (C50), dynamic range, or contrast dominance. In sum, the overall similarity between current and voltage responses indicates that voltage-sensitive conductances do not play a major role in determining the shape of the bipolar cell's contrast response in the light-adapted retina. The salient characteristics of the contrast response of bipolars apparently arise between the level of the cone voltage response and the postsynaptic current of bipolar cells, via the transformation between cone voltage and transmitter release and/or via the interaction between the neurotransmitter glutamate and its postsynaptic receptors on bipolar cells.

Intracellular recordings were obtained from 73 cone-driven bipolar cells in the light-adapted retina of the tiger salamander (Ambystoma tigrinum). Responses to flashes of negative and positive contrast for centered spots and concentric annuli of optimum spatial dimensions were analyzed as a function of contrast magnitude. For both depolarizing and hyperpolarizing bipolar cells, it was found that remarkably similar responses were observed for the center and surround when comparisons were made between responses of the same response polarity and thus, responses to opposite contrast polarity. Thus, spatial information and contrast polarity appear to be rather strongly confounded in many bipolar cells. As a rule, the form of the contrast/response curves for center and surround approximated mirror images of each other. Contrast gain and C50 (the contrast required for half-maximal response) were quantitatively similar for center and surround when comparisons were made for responses of the same response polarity. The average contrast gain of the bipolar cell surround was 3–5 times higher than that measured for horizontal cells. Contrast/latency measurements and interactions between flashed spots and annuli showed that the surround response is delayed by 20–80 ms with respect to that of the receptive-field center. Cones showed no evidence for center-surround antagonism while for bipolar cells, the average strength of the surround ranged from about 50% to 155% of the center, depending on the test and response polarity. The results of experiments on the effects of APB (100 μM) on depolarizing bipolar cells suggest that the relative contribution of the feedback pathway (horizontal cell to cones) and the feedforward pathway (horizontal cell to bipolar cell) to the bipolar surround varies in a distributed manner across the bipolar cell population.

Effects of light adaptation on contrast processing in the outer retina were investigated over nearly four decades of background illumination by analyzing the intracellular responses of 111 bipolar cells, 66 horizontal cells, and 22 cone photoreceptors in the superfused eyecup of the tiger salamander (Ambystoma tigrinum). Light adaptation had striking and similar effects on the average contrast responses of the hyperpolarizing (Bh) and depolarizing (Bd) classes of bipolar cells: Over the lower two decades of background illumination, the contrast gain increased 7-fold to reach values as high as 20–30, the dynamic range and the half-maximum contrast decreased by about 60%, the total voltage range increased some 40%, and contrast dominance changed from highly positive to more balanced. At higher levels of background, most aspects of the contrast response stabilized and Weber's Law then held closely. In this background range, the contrast gain of bipolar cells was amplified some 20× relative to that of cones whereas the corresponding amplification in horizontal cells was about 6×. Differences in the growth of contrast gain with the intensity of the background illumination for cones versus bipolar cells suggest that there are at least two adaptation-dependent mechanisms regulating contrast gain. One is evident in the cone photoresponse such that an approximately linear relation holds between the steady-state hyperpolarization and contrast gain. The other arises between the voltage responses of the cones and bipolar cells. It could be presynaptic (modulation of cone transmitter release by horizontal cell feedback or other mechanisms) and/or postsynaptic, that is, intrinsic to bipolar cells. Contrast gain grew with the background intensity by a larger factor in horizontal than in bipolar cells. This provides a basis for the widely held view that light adaptation increases the strength of surround antagonism in bipolar cells. On average, the effects of light adaptation and most quantitative indices of contrast processing were remarkably similar for Bd and Bh cells, implying that both classes of bipolar cells, despite possible differences in underlying mechanisms, are about equally capable of encoding all primary aspects of contrast at all levels of light adaptation.

The impulse discharge of single ganglion cells was recorded extracellularly in superfused eyecup preparations of the tiger salamander (Ambystoma tigrinum). Contrast flashes (500 ms) were applied at the center of the receptive field while the retina was light adapted to a background field of 20 cd/m2. The incidence of cell types in a sample of 387 cells was: ON cells (4%), OFF cells (28%), and ON/OFF cells (68%). Quantitative contrast/response measurements were obtained for 83 cells. On the basis of C50, the contrast necessary to evoke a half-maximal response, ON/OFF cells fell into 3 groups: (1) Positive Dominant (26%), (2) Balanced (23%), and (3) Negative Dominant (51%). Positive Dominant cells tended to be relatively contrast insensitive. On the other hand, many Negative Dominant cells showed remarkably low C50 values and very steep contrast/response curves. Contrast gain to negative contrast averaged 8.5 impulses/s/% contrast, some four times greater than that evoked by positive contrast. In most ON/OFF cells, the latency of the first spike evoked by a negative contrast step was much shorter (40–100 ms) than that evoked by a positive contrast step of equal contrast. OFF cells typically showed higher C50 values, larger dynamic ranges, and longer latencies than those of Negative Dominant ON/OFF cells. Thus, different pathways or mechanism apparently mediate the off responses of OFF and ON/OFF cells. In sum, the light-adapted retina of the tiger salamander is strongly biased in favor of negative contrast, as shown by the remarkably high contrast sensitivity and faster response of Negative Dominant cells, the remarkably low incidence of ON cells, and the insensitivity of Positive Dominant cells. Some possible underlying influences of bipolar and amacrine cells are discussed.