We examined the mechanisms underlying the generation of the surround response in cone photoreceptors in retinal slice preparations of the newt. Under current-clamp recording, a surround response appearing at around −20 mV was suppressed by both depolarization and hyperpolarization of the membrane voltage, which is consistent with a number of previously published reports that have suggested that the surround response of the cone photoreceptors is voltage dependent (turtle, O'Bryan, 1973
; tiger salamander cones, Skrzypek and Werblin, 1983
; Wu, 1991
). Current flow through gap junctions between neighboring cones (DeVries et al., 2002
) did not generate a surround response in the present study, because the amplitude of the surround response was independent of the difference between the membrane voltage and the presumed resting potential of neighboring cones. In the case of voltage-clamp recording, the surround response appeared in the voltage range in which the cone ICa
was activated, which indicates that the surround response of the cones is closely related to ICa
. The contribution of cone ICa
to the surround response has been demonstrated previously in turtle (Piccolino and Gerschenfeld, 1978
; Gerschenfeld and Piccolino, 1980
; Thoreson and Burkhardt, 1991
) and goldfish (Verweij et al., 1996
; Kraaij et al., 2000
) retinas. In this study, we focused on the mechanism by which surround illumination triggers activation of cone ICa
Feedback Mediated by pH Changes in the Outer Retina
Several factors have been proposed as candidate feedback messengers from the HCs to cones. These include substances that modulate ICa
of the cones, such as nitric oxide, glutamate, chloride, protons, and the ephaptic effect caused by current flowing into the HCs (for review see Kamermans and Spekreijse, 1999
). Enrichment of the pH buffering capacity of the extracellular solution increased cone ICa
and prevented any additional increase in ICa
produced by surround illumination. Thus, we consider that protons are the most likely candidates among these substances. Moreover, HC depolarization by kainate suppressed cone ICa
, whereas HC hyperpolarization by CNQX increased it. Therefore, it is highly plausible that the pH in the cone synaptic cleft is tightly controlled by the membrane voltage of the HCs.
The most plausible interpretation for the effects of enrichment of the pH buffering capacity is that the surround response of the cones is modulated by pH changes in the invaginating synaptic clefts, and thus the following sequences are considered. In a normal bicarbonate-buffer solution, pH in the synaptic cleft is slightly more acidic (by about pH 0.2; estimated in discussion) than the pH in the large extracellular pool (pH 7.4). Thus, cone ICa is relatively suppressed. The HC hyperpolarization caused by surround illumination neutralizes this acidic condition, thus restoring cone ICa and the cone surround response. On the other hand, in an external solution with a high buffering capacity, pH in the synaptic cleft is already fixed to the same pH as that of the extracellular pool (pH 7.4) even in darkness, and thus the enhancement of cone ICa by surround illumination does not occur.
The assumed pH change of 0.2 U may be a large enough change for pH electrode to detect. Therefore, we measured the pH in the outer plexiform layer by inserting a pH-selective microelectrode (tip diameter: 2 μm) into the outer synaptic region in the retinal slices. The fabrication method of the microelectrode used was that described by Smith et al. (1999)
and Molina et al. (2000)
. The smallest pH change that our pH micropipette could detect was 0.06 pH U. However, no pH changes were detected after the addition of kainate (synaptic clefts should be acidified) and CNQX (the clefts should be alkalinized) to the superfusate in 23 slices examined. However, the absence of detectable pH change may not mean that the pH of the synaptic clefts is fixed. Insertion of the pH-sensitive micropipette may open up the narrow space at the invaginating synapse and make a wide artificial channel to the bath of the recording chamber. Through this artificial channel, any local pH change, that might be large enough within a narrow space, may become diluted and become undetectable.
Recently, DeVries (2001)
demonstrated that exocytosed protons from the cone terminal mediate a feedback to block the cone ICa.
The exocytosed protons can form a negative feedback loop to control glutamate release in a sustained manner. In contrast, synaptic acidification caused by HC depolarization can depend on the illumination area of the surround light, and thus contribute to receptive field surround formation in the outer retina.
If the pH in the synaptic cleft is controlled by the membrane voltage of the HCs, the mechanism by which surround illumination augmented the cone ICa
can be interpreted easily. Kamermans et al. (2001)
proposed hemichannel-mediated ephaptic feedback in the goldfish retina, based on following evidence. (a) The presence of hemichannel proteins in the dendrite tips of the HCs, and (b) the blocking effect of carbenoxolone, a blocker of hemichannels, on the feedback responses. If the ephaptic effect modulates cone ICa
, surround illumination should be expected to shift the I-V curve of the cone ICa
at all voltages (like in B b). However, importantly, surround illumination never induced a parallel shift of the I-V curve of the cone ICa
in the negative direction; rather it increased ICa
even in the region of the positive slope of the I-V curve of the cone ICa
(between −10 and 10 mV; B a), which is inconsistent with the ephaptic feedback hypothesis.
Protonated aminosulfonate compounds, including HEPES, have been reported to decrease the permeability of connexin-26 hemichannels, but compounds without an aminosulfonate moiety, such as Tris, maleate, and bicarbonate, do not decrease the permeability of these channels (Bevans and Harris, 1999
). The effects of HEPES on the surround response are not likely to be due to blockade of the connexin-26 hemichannels in HCs (Kamermans et al., 2001
), because Tris also suppressed the surround responses. Furthermore, there are other phenomena which seem to lend greater support to the pH-feedback hypothesis. First, in five of eight cones, 10 mM HEPES partially blocked the surround response at −26 mV (inset in B b). Since cone ICa
was most sensitive to external pH change at −26 mV (), even a slight change in pH was detectable. Second, when large surround responses of 20 pA were recorded in the control solution, diffuse light occasionally elicited a current dip at the offset of illumination (but not at the beginning) in the enriched buffer solution ( A, arrow). This may be interpreted to mean that the rate of proton buffering by Tris did not match up well with the rate of the sudden increment of protons at the time of switch-off of the surround light.
In this study, surround illumination shifted the activation curve of the cone ICa
by ~2.5 mV, which corresponded to the alkalinization-mediated shift of pH by ~0.2 (estimated from Barnes et al., 1993
) in a pH 7.4 external solution. In retinal slices, the surround response may be weakened due to the reduction of the receptive field size. Thus, the pH change by surround illumination in the retinal slice may be an underestimate of the value prevailing in vivo. In an isolated retina of the goldfish, surround illumination shifted the I-V curve of the cone ICa
by 7.5 mV (Verweij et al., 1996
), which corresponds to an alkalinization of ~0.7 pH U. It is possible that the surround illumination induced alkalinization by 0.2 pH U in the bicarbonate buffer solution. In the retina, light stimulation evokes alkalinization of up to 0.2 pH U in the intraretinal extracellular space (Yamamoto et al., 1992
), probably due to the change of H+
release caused by the energy metabolism in retinal cells. The activity-dependent changes in the external pH can play an active role in neuronal activity, as well as in the basal metabolism of retinal systems.
External protons also inhibit the glutamate response of AMPA receptors by increasing steady-state desensitization (Ihle and Patneau, 2000
). However, the IC50
values for proton inhibition exceed the physiological range (from pH 5.7 to 6.3; Traynelis and Cull-Candy, 1991
). Thus, during the generation of the cone surround response, pH changes in the cone synaptic clefts may affect the presynaptic cone calcium channels selectively, but not the postsynaptic AMPA receptors on the HCs.
In off-type BCs, 10 mM HEPES augmented the inward current response at the offset of a light spot, in addition to suppressing the surround response (). This is probably due to augmentation of transmitter release from the photoreceptors, and is consistent with the fact finding that HEPES augmented the cone ICa
(). The sustained inward current in darkness, however, was not significantly augmented and this was probably due to desensitization of postsynaptic kainate receptors on off-BCs (DeVries and Schwartz, 1999
). Under spot illumination (glutamate release is stopped), the kainate receptors would be released from a desensitized state. Thus, at light offset, the postsynaptic current evoked by abrupt glutamate release can be marked. A picrotoxin-resistant surround response of off-type BCs has also been reported in the tiger salamander retina (Hare and Owen, 1996
). Thus, pH-mediated feedback to cones, rather than a GABAergic feedforward input to BCs, may be mainly involved in the receptive field organization of the BCs, at least in the amphibian retina. Modulation of the cone ICa
is very strategic for direct control of the amount of transmitter release. Thus, the modulatory effect of pH on ICa
at the cone synaptic terminal has a dual role, of contributing to the formation of the receptive field surround in the cones, and of controlling transmitter release from the cones to the BCs.
A Possible Mechanism of Control of the Extracellular pH Associated with Voltage Change in the HCs
There are several reports that neuronal activities evoke pH changes in the extracellular spaces in neuronal tissue (Kaila and Chesler, 1998
). However, the mechanism by which HC depolarization acidifies the extracellular space in the cone synaptic clefts remains to be elucidated. An applicable model for the present hypothesis has been proposed as the model of neuron–glial cell interaction (Ransom, 2000
). In this model, depolarization of glial cells caused by neuronal excitation activates Na+
cotransport (stoichiometry of 2HCO3−
) in the glia, resulting in the cellular uptake of extracellular HCO3–
and acidification of the extracellular space (Ransom, 2000
). If extracellular protons are increased in darkness, the extracellular space in a tightly packed invaginating synapse might be easily acidified. Also in isolated HCs of the skate retina, Na+
- and HCO3−
-dependent ion transport regulates the intracellular pH (Haugh-Schmedt and Ripps, 1998
). Besides Na+
cotransport in HCs, other ionic transporters or exchangers, such as acid loaders or extruders must also be considered. Moreover, proton release via vesicular release of GABA, which has been suggested in mammalian HCs (Cueva et al., 2002
), must also be considered.
Molina et al. (2000)
reported that application of glutamate to isolated HCs of the all-rod skate retina reduces the number of hydrogen ions on its surface, indicating that glutamate causes proton influx into the cytoplasm of the HCs. This glutamate-mediated proton influx into the HCs is consistent with the evidence that L-glutamate raises the intracellular proton concentration in isolated HCs (Dixon et al., 1993
). The results of Molina et al. (2000)
and Dixon et al. (1993)
appear to be inconsistent with our conclusion that the extracellular space should be acidified in darkness (when glutamate is tonically released). However, since the experiments by Molina et al. (2000)
and Dixon et al. (1993)
were performed with an external solution buffered with HEPES (without bicarbonate), the bicarbonate-dependent system may not be applicable in their studies. Moreover, Molina et al. (2000)
performed their study using rod-driven HCs, whereas our discussion is on pH regulation in cone systems. Therefore, these two studies are not necessarily inconsistent with our present results obtained using bicarbonate buffer in the external solution.
Contribution of GABAergic Feedback to Receptive Field Organization
Our present analysis was focused on GABA-independent components; therefore, all recordings were made in the presence of 100 μM picrotoxin, to exclude any possible effects of GABA. It has been a matter of debate whether GABA is also involved in the formation of the receptive field surround of cone photoreceptors. Our observations indicate clearly that there is a large component that does not require GABA. In fact, in preliminary studies, we found that the amplitude of cone surround response was not reduced by GABA antagonists (picrotoxin, bicculine, SR95531). Moreover, the GABA antagonists did not evoke any current change whose reversal potential was equal to the equilibrium potential of chloride ions (unpublished data). Thus, it is unlikely that GABA plays a major role as a mediator of the feedback in the outer retina.
GABA may, however, play a substantial role in modulating the feedback response in the outer retina, rather than being the main mediator of the feedback. GABA can change membrane voltages of the HCs by modulating GABA-gated chloride currents and GABA transporter currents in the HCs (Kamermans and Werblin, 1992
; Takahashi et al., 1995a
), besides modulating GABA-gated chloride currents in cones. Thus, it is assumed that GABA may play some role in the information processing in the outer retina, but its role has to be reexamined in light of new data on the surround response of cones.
The synaptic structure of the invaginating synapse may be specialized for evoking pH changes in the intersynaptic clefts, because its highly packed structure can promote accumulation of protons. In contrast, GABAergic synapses between amacrine cells and bipolar and ganglion cells are conventional synapses, much different from the invaginating type of synapse. Thus, it may be reasonable to assume that lateral inhibition in the inner retina is mediated by GABA (Cook and McReynolds, 1998
In summary, we have proposed an alternative hypothesis for the mechanism underlying the generation of the receptive field surround in the outer retina. We propose that intersynaptic pH changes in the invaginating synaptic clefts at the cone terminal contribute to the generation of the receptive field surround in the outer retina. Although the mechanism by which HCs regulate the extracellular pH is still uncertain, the present pH hypothesis should serve as the simplest description, so far, for the mechanism of feedback in the outer retina.