Hajime Hirasawa

pH Changes in the Invaginating Synaptic Cleft Mediate Feedback from Horizontal Cells to Cone Photoreceptors by Modulating Ca2+ Channels

Feedback from horizontal cells (HCs) to cone photoreceptors plays a key role in the center-surround–receptive field organization of retinal neurons. Recordings from cone photoreceptors in newt retinal slices were obtained by the whole-cell patch-clamp technique, using a superfusate containing a GABA antagonist (100 μM picrotoxin). Surround illumination of the receptive field increased the voltage-dependent calcium current (ICa) in the cones, and shifted the activation voltage of ICa to negative voltages. External alkalinization also increased cone ICa and shifted its activation voltage toward negative voltages. Enrichment of the pH buffering capacity of the extracellular solution increased cone ICa, and blocked any additional increase in cone ICa by surround illumination. Hyperpolarization of the HCs by a glutamate receptor antagonist-augmented cone ICa, whereas depolarization of the HCs by kainate suppressed cone ICa. From these results, we propose the hypothesis that pH changes in the synaptic clefts, which are intimately related to the membrane voltage of the HCs, mediate the feedback from the HCs to cone photoreceptors. The feedback mediated by pH changes in the synaptic cleft may serve as an additional mechanism for the center-surround organization of the receptive field in the outer retina.

GABA-mediated component in the feedback response of turtle retinal cones.

The negative feedback from horizontal cells to cone photoreceptors contributes to the formation of the receptive-field surround in cone photoreceptors. Recently, studies on the modulation of voltage-gated Ca(2+) currents in cone photoreceptors have led to great progress in our understanding of the mechanism of horizontal-cone feedback. Another highly probable hypothesis is that GABA mediates this feedback. This hypothesis is supported by the facts that cone photoreceptors respond to GABA and that horizontal cells release GABA. However, GABA-mediated synaptic inputs from horizontal cells to cone photoreceptors have not been demonstrated. In the present study, we examined whether cone photoreceptors receive GABAergic inputs from horizontal cells using a slice patch technique in the turtle retina. When 1 mM of GABA was applied to the cone photoreceptors, GABA-induced currents were activated. GABA-induced currents reversed their polarity at the equilibrium potential of Cl-. The application of 30 microM of SR95531, an antagonist of GABAA receptors, alone did not produce any change in the holding currents. When 200 microM of pentobarbital was introduced to potentiate the GABAergic inputs to the cone photoreceptors, however, the inhibitory action of SR95531 on GABAergic inputs became detectable. The amplitude of the GABAergic inputs, potentiated by pentobarbital, increased when the horizontal cells were depolarized by the application of 20 microM of kainate, while the amplitude decreased when the horizontal cells were hyperpolarized by the application of 10 microM of CNQX. When the cone photoreceptors were voltage clamped at a potential at which the voltage-gated Ca(2+) current was inactive, horizontal-cone feedback was not observed. However, the horizontal-cone feedback became detectable when the GABAergic inputs to the cone photoreceptors were potentiated by pentobarbital. We concluded that the contribution of GABAergic inputs from horizontal cells to cone pedicles in the formation of the receptive-field surround in cone photoreceptors is very limited but that the modulation of voltage-gated Ca(2+) currents in cone photoreceptors is a physiologically relevant mechanism for horizontal-cone feedback.

Expression of circadian clock genes in retinal dopaminergic cells.

The mammalian neural retina contains single or multiple intrinsic circadian oscillators that can be directly entrained by light cycles. Dopaminergic amacrine (DA) cells represent an especially interesting candidate as a site of the retinal oscillator because of the crucial role of dopamine in light adaptation, and the widespread distribution of dopamine receptors in the retina. We hereby show by single-cell, end-point RT-PCR that retinal DA cells contain the transcripts for six core components of the circadian clock: Bmal1, Clock, Cry1, Cry2, Per1, and Per2. Rod photoreceptors represented a negative control, because they did not appear to contain clock transcripts. We finally confirmed that DA cells contain the protein encoded by the Bmal1 gene by comparing immunostaining of the nuclei of DA cells in the retinas of wildtype and Bmal1-/- mice. It is therefore likely that DA cells contain a circadian clock that anticipates predictable variations in retinal illumination.

Corelease of Dopamine and GABA by a Retinal Dopaminergic Neuron

Numerous neurons release two transmitters of low molecular mass, but it is controversial whether they are localized within the same synaptic vesicle, with the single exception of GABA and glycine because they are ferried into the vesicle by the same transporter. Retinal dopaminergic (DAergic) amacrine cells synthesize both dopamine (DA) and GABA. Both transmitters are released over the entire cell surface and act on neighboring and distant neurons by volume transmission, but, in addition, DAergic cells establish GABAergic synapses onto AII amacrine cells, the neurons that transfer rod signals to cone bipolars. By combining recordings of DA and GABA release from isolated, genetically identified perikarya of DAergic cells from the mouse retina, we observed that a proportion of the events of DA and GABA exocytosis were simultaneous, suggesting corelease. Furthermore, a proportion of the secretory organelles in the perikaryon and synaptic endings of DAergic cells contained both vesicular transporters for DA [vesicular monoamine transporter 2 (VMAT2)] and GABA [vesicular GABA transporter (VGAT)]. Because the majority of the DA release events concerned a single transmitter and organelles were present that contained a single transporter, either VMAT2 or VGAT, we conclude that the secretory organelles of DAergic cells contain variable concentrations of the two transmitters, which are in turn determined by a variable mixture of the two transporter molecules in their limiting membrane. This variability can be explained if the relative numbers of transporter molecules is determined stochastically during the budding of the somatic organelles from the trans-Golgi network or the retrieval of the vesicular membrane from the plasmalemma after exocytosis.

Acidification of the synaptic cleft of cone photoreceptor terminal controls the amount of transmitter release, thereby forming the receptive field surround in the vertebrate retina.

In the vertebrate retina, feedback from horizontal cells (HCs) to cone photoreceptors plays a key role in the formation of the center-surround receptive field of retinal cells, which induces contrast enhancement of visual images. The mechanism underlying surround inhibition is not fully understood. In this review, we discuss this issue, focusing on our recent hypothesis that acidification of the synaptic cleft of the cone photoreceptor terminal causes this inhibition by modulating the Ca channel of the terminals. We present evidence that the acidification is caused by proton excretion from HCs by a vacuolar type H+ pump. Recent publications supporting or opposing our hypothesis are discussed.

Blocking AMPA receptor desensitization prolongs spontaneous EPSC decay times and depolarizes H1 horizontal cells in carp retinal slices

Desensitization of H1 horizontal cell (H1 HC) glutamate receptors was investigated in carp retinal slices using cyclothiazide (CTZ), an inhibitor of AMPA receptor desensitization. 100 microM CTZ depolarized H1 HCs and increased the amplitude of light responses, without any prominent changes in their kinetics. Spontaneous EPSCs (sEPSCs) in H1 HCs were observed in the presence of 2.5 mM heptanol, an uncoupling agent of gap junctions. 20 microM GYKI52466 (an AMPA receptor antagonist) blocked the sEPSCs, consistent with the sEPSCs being mediated by AMPA receptors. 100 microM cobalt suppressed the frequency of sEPSCs without changing their mean peak amplitude, suggesting that calcium-dependent transmitter release from cones was not affected by heptanol. CTZ increased the total inward charge transferred per sEPSC by increasing the sEPSC decay time constant twofold, without any significant change in their frequency and mean peak amplitude. This suggests that the depolarizing effect of CTZ on H1 HCs was due to blocking desensitization of AMPA receptors, increasing the inward current induced by glutamate released from cone synaptic terminals. The desensitization of glutamate receptors may function to extend the dynamic range of H1 HC light responses.

Analysis of spontaneous EPSCs in retinal horizontal cells of the carp

Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded under Whole-cell voltage clamp from carp type 1 horizontal cells (H1 cells) uncoupled by dopamine in retinal slices. Red light steps, which hyperpolarise cones and reduce glutamate release, induced outward current responses accompanied by a suppression of sEPSCs. sEPSCs decayed exponentially with a mean time constant of 0.71+/-0.07 ms and had a reversal potential near 0 mV. Power spectral analysis of sEPSCs revealed a similar decay time constant. They were suppressed by a non-NMDA receptor antagonist, CNQX at 10 microM, and a relatively specific AMPA receptor antagonist, GYKI52466 at 20 microM. The presence of sEPSCs suggests that the release of glutamate from cone synaptic terminals is vesicular. The reduction in mean sEPSC frequency with red light was not accompanied by a significant change in the mean sEPSC conductance increase (482+/-59 pS), suggesting that a decrease in the vesicular release rate from cones does not alter the vesicular glutamate concentration (quantal contents). The results suggest that the spontaneous events in H1 cells were contributed by non-NMDA (possibly AMPA) type glutamate receptors modulated by the red cone input.

Effects of nitric oxide, light adaptation and APB on spectral characteristics of H1 horizontal cells in carp retina

The spectral characteristics of cone-driven horizontal cells of H1 subtype (H1 HCs) receiving main synaptic input from red-sensitive cones were studied in light- and dark-adapted retinae of carp. The spectral sensitivity profile of H1 HCs in dark-adapted retinae was practically the same as the absorption spectrum of red-sensitive cones. Light-adaptation decreased the sensitivity preferentially in the short-wavelength (blue/green) region, resulting in a relative enhancement of the 617 nm peak. Application of nitric oxide (NO) donors, sodium nitroprusside (SNP) and S-nitrosoglutathione (SNOG or GSNO), or dopamine to dark-adapted retinae decreased the sensitivity preferentially in blue/green region, an effect similar to that of light-adaptation. Application of haemoglobin (Hb, an NO scavenger) or 2-amino-4-phosphonobutyrate (APB, a metabotropic glutamate receptor agonist), to light-adapted retinae increased the sensitivity preferentially in the blue/green region, an effect similar to dark-adaptation. The photoresponses of H1 HCs were univariant in dark-adapted retinae as well as Hb-treated light-adapted retinae. In light-adapted retinae with normal Ringer, however, the univariance did not hold. These results suggested that the photoresponses of H1 HCs to short-wavelength stimuli contain a depolarising (sign-reversing) component, which can be activated by light-adaptation or application of NO and dopamine, and inactivated by dark-adaptation or deprivation of NO or application of APB.

External Proton Mediates the Feedback from Horizontal Cells to Cones in the Newt Retina

The center-surround organization of the receptive fields is one of the most important properties of retinal neurons. Cone photoreceptors show concentric receptive field; they are hyperpolarized by spot light and depolarized by annulus light. It is widely accepted that the negative feedback from horizontal cells (HCs) generates the surround response of cones. Although accumulating evidence suggests that γ-aminobutyric acid (GABA) mediates the feedback from HC to cones, there are still a number of reports criticizing the GABA hypothesis of HC-cone feedback. In the goldfish, Verweij et al. [1] have reported that the feedback was detectable in the presence of GABA antagonist, and the surround illumination shifted the activation voltage of the cone calcium current (ICa) to be more negative. In the present study, we reexamined the mechanism of feedback to find an answer to this controversy.

Analysis of the Proton Mediated Feedback Signals in the Outer Plexiform Layer of Goldfish Retina

Center-surround antagonistic receptive field in the retina is generated by negative feedback from horizontal cells (HCs) via a proton feedback mechanism [1]. In this study, the contribution of protons on the color opponent signal formation is analyzed. Increasing the buffer capacity of the external medium by 10 mM HEPES depolarized the dark membrane potential of HCs, and substantially increased hyperpolarizing responses to light stimulation. In contrast, feedback mediated depolarizing responses of H2 and H3 HCs were suppressed by HEPES. Moreover, depolarizing response onset of H2 and H3 HCs was significantly delayed compared to the hyperpolarizing responses. These indicate that proton plays an important role on the color opponent signal formation of HCs, and that the feedback from H1 to H2 HCS is delayed by 10 – 20 ms. A similar delay might be applicable to other feedback pathways as well.