Synaptic currents generating the inhibitory surround of ganglion cells in the mammalian retina

Abstract

The receptive field (RF) of retinal ganglion cells (RGCs) consists of an excitatory central region, the RF center, and an inhibitory peripheral region, the RF surround. It is still unknown in detail which inhibitory interneurons (horizontal or amacrine cells) and which inhibitory circuits (presynaptic or postsynaptic) generate the RF surround. To study surround inhibition, light-evoked whole-cell currents were recorded from RGCs of the isolated, intact rabbit retina. The RFs were stimulated with light or dark spots of increasing diameters and with annular light stimuli. Direct inhibitory currents could be isolated by voltage clamping ganglion cells close to the Na(+)/K(+) reversal potential. They mostly represent an input from GABAergic amacrine cells that contribute to the inhibitory surround of ganglion cells. This direct inhibitory input and its physiological function were also investigated by recording light-evoked action potentials of RGCs in the current-clamp mode and by changing the intracellular Cl(-) concentration. The excitatory input of the ganglion cells could be isolated by voltage clamping ganglion cells at the Cl(-) reversal potential. Large light spots and annular light stimuli caused a strong attenuation of the excitatory input. Both GABA(A) receptors and GABA(C) receptors contributed to this inhibition, and picrotoxinin was able to completely block it. Together, these results show that the RF surround of retinal ganglion cells is mediated by a combination of direct inhibitory synapses and presynaptic surround inhibition.

Fig. 1.

Drawings of four ganglion cells from whole-mounted rabbit retinas, viewed from the ganglion cell side. The cells were filled with Neurobiotin during patch-clamp recordings. The axons are indicated by the arrows, and the horizontal axis is parallel to the visual streak. A, α ganglion cell from the center of the streak. Recordings from this cell are shown in Figure6. B, δ-Like ganglion cell from an eccentricity (distance from the center of the streak) of 800 μm. Recordings from this cell are shown in Figure 8. C, Bistratified ON–OFF direction-selective ganglion cell. The solid dendrites stratify in the inner IPL, and the dotted dendrites branch 9 μm farther, toward the outer IPL. The cell was from an eccentricity of 200 μm. D, Sluggish concentric ganglion cell from an eccentricity of 1.2 mm. Recordings from this cell are shown in Figures 2A and 5. Scale bar, 100 μm.

Fig. 2.

Light-induced currents of retinal ganglion cells voltage clamped at V_H of −75 mV.A, This ON ganglion cell (GC) was stimulated with different sized light spots of 400 msec duration (top trace). Their diameters (in micrometers) are indicated on the five current traces. B, This OFF ganglion cell (GC) was stimulated with dark spots of different sizes (diameters as in A). C, Area–response function of the ON ganglion cell shown inA. The abscissa shows the diameters of the light spots, and the ordinate shows the normalized responses of the cell. The peak amplitudes of the currents, their sustained components, and the charge transfer (integral of the current over the time axis) were measured. D, Area–response function of the OFF ganglion cell shown in B.

Fig. 3.

Reversal potentials of the light-induced currents of an ON–OFF ganglion cell. A, The cell was voltage clamped at different holding potentials (V_HOLD; shown on theleft), and the light-induced currents are shown. Light spots of 50, 200, and 1000 μm diameter and an annulus (inner diameter 200 μm, outer diameter 1000 μm) were projected into the receptive field (top trace). Calibration: 400 msec, 100 pA. Peak currents were measured as the average current between the two solid lines, and sustained currents were measured as the average current between the two dotted lines. B, Current–voltage curves of the peak currents measured in A for the different light stimuli. C, Current–voltage curves of the sustained currents measured inA for the different light stimuli.

Fig. 4.

A, Increase of the light response of an ON center ganglion cell with increasing light intensity. The cell was voltage clamped at V_H of −75 mV. Theordinate shows the charge transfer into the cell in picocoulombs (integral of the current over the 400 msec of the light stimulus), and the two curves show the intensity–response functions for two spot sizes. Theabscissa shows the light intensity in relative units. The intensity 1000 represents 0.7 cd/m² at the monitor. B, Reversal potentials of the light-induced currents of this ganglion cell for light spots of 100 μm (open symbols) and 1200 μm (filled symbols) diameter. The curves were measured at three different intensities, and the sustained currents are shown. The reversal potentials are independent of the light intensities.

Fig. 5.

A, Light-induced currents of an ON ganglion cell (same cell as Fig. 1A) that was voltage clamped at three different holding potentials. The holding potential V_H of 0 mV represents the Na⁺/K⁺ reversal potential, andV_H of −55 mV is the Cl⁻reversal potential. B, Area–response functions of the light-induced currents in A, measured atV_H of −55 mV (excitatory current) andV_H of 0 mV (inhibitory current).

Fig. 6.

Light-induced action potentials of an ON ganglion cell recorded in the current-clamp mode. The records inA were performed with an electrode containing a low Cl⁻ concentration. The light stimuli were a spot of 400 μm diameter (top trace), an annulus of inner diameter 400 μm and outer diameter 1200 μm (middle), and a large spot of 1200 μm diameter. The records in Bwere taken from the same cell with an electrode containing a high Cl⁻ concentration. Same light stimuli as inA were used.

Fig. 7.

Light-induced currents of ganglion cells that were voltage clamped at V_H of 0 mV, the Na⁺/K⁺ reversal potential. The cells were stimulated with an annulus. A, Application of bicuculline and strychnine to the bathing medium caused a substantial reduction of the outward currents recorded from this ON–OFF ganglion cell. The time after the drug application is shown on thetraces. B, Application of picrotoxinin to the bathing medium completely blocked the outward current of this ON ganglion cell.

Fig. 8.

Light-induced currents of an OFF ganglion cell that was voltage clamped at the Cl⁻ reversal potential (V_H of −45 mV). A, Spots of increasing diameters elicited transient inward currents that were strongly attenuated with large spots. B, Application of picrotoxinin (100 μm) caused a substantial increase of the light-evoked currents and became more sustained, and large spots did not attenuate the currents. C, Area–response curves showing the charge transfer (in picocoulombs) of the currents in A andB, respectively. D, Normalized area–response curves of the records in A andB, respectively. In the control record, the large spot attenuation is apparent, and during application of picrotoxinin this attenuation appears to be primarily blocked.

Fig. 9.

TTX application reduces lateral inhibition.A, Light-induced currents of an ON ganglion cell that was voltage clamped at V_H of 0 mV and stimulated with an annulus (inner diameter 300 μm, outer diameter 1000 μm). The sustained outward current was blocked by the application of TTX. B, Area–response curves of another ON ganglion cell that was voltage clamped atV_H of −45 mV. The light responses in the control recordings show a strong attenuation for large spots. When TTX was applied, this attenuation was substantially reduced.