Action potentials are required for the lateral transmission of glycinergic transient inhibition in the amphibian retina

Abstract

Transient lateral inhibition (TLI), the suppression of responses of a ganglion cell to light stimuli in the receptive field center by changes in illumination in the receptive field surround, was studied in light-adapted mud puppy and tiger salamander retinas using both eyecup and retinal slice preparations. In the eyecup, TLI was measured in on-off ganglion cells as the ability of rotating, concentric windmill patterns of 500-1200 micron inner diameter to suppress the response to a small spot stimulus in the receptive field center. Both the suppression of the spot response and the hyperpolarization produced in ganglion cells by rotation of the windmill were blocked in the presence of 2 microM strychnine or 500 nM tetrodotoxin (TTX), but not by 150 microM picrotoxin. In the slice preparation in which GABA-mediated currents were blocked with picrotoxin, IPSCs elicited by diffuse illumination were blocked by strychnine and strongly reduced by TTX. The TTX-resistant component was probably attributable to illumination of the receptive field center. TTX had a much greater effect in reducing the glycinergic inhibition elicited by laterally displaced stimulation versus nearby focal electrical stimulation. Strychnine enhanced light-evoked excitatory currents in ganglion cells, but this was not mimicked by TTX. The results suggest that local glycinergic transient inhibition does not require action potentials and is mediated by synapses onto both ganglion cell dendrites and bipolar cell terminals. In contrast, the lateral spread of this inhibition (at least over distances >250 micron) requires action potentials and is mainly onto ganglion cell dendrites.

Fig. 1.

Strychnine blocks TLI in ganglion cells. Responses are from an on–off ganglion cell in tiger salamander eyecup. Each trace is the average of four to eight responses that had been filtered to eliminate action potentials (see Methods and Materials). Each of the three groups of traces shows the average response to a 400-μm-diameter spot alone (left) and in the presence of a windmill pattern (1200 μm i.d., 2600 μm o.d.) that was either stationary (middle) or rotating (right). The timing of the spot and windmill stimuli are indicated by thehorizontal bars at the bottom. Spot and windmill intensity were both 8.4 log quanta. Responses in themiddle row of traces were obtained after 5 min in 2 μm strychnine, and those in the bottom rowof traces were obtained 20 min after return to control Ringer’s solution. Resting potential in darkness was −50 mV in control Ringer’s solution and −53 mV in the presence of strychnine.

Fig. 2.

TTX blocks TLI in ganglion cells. Responses are from the same cell as in Figure 1. Except for the use of TTX rather than strychnine, all conditions are the same as in Figure 1. The responses in the middle row of traces were obtained after 2 min in 500 nm TTX, and those in the bottom row of traces were obtained 10 min after return to control Ringer’s solution. Resting potential was −51 mV in both control Ringer’s solution and TTX.

Fig. 3.

Summary of effects of strychnine, picrotoxin, and tetrodotoxin on transient lateral inhibition in ganglion cells. TLI was elicited by 1200 μm i.d. windmill stimuli, intensity 8.4 log quanta. Ordinate indicates the magnitude of TLI expressed as percentage suppression of the spot response by rotation of the windmill pattern, calculated as described in Materials and Methods. Each pair ofbars shows TLI in control Ringer’s solution (open) and in the presence of the indicated drug (hatched): 2 μm strychnine (STR) (n = 6 cells), 150 μm picrotoxin (PTX) (n = 3 cells), and 500 nm tetrodotoxin (TTX) (n = 6 cells). In three of the cells both strychnine and TTX were tested. Error bars indicate 1 SEM. Strychnine and TTX each caused a significant decrease in TLI (p = 0.004 and 0.003, respectively; paired Student’s t test), but picrotoxin did not (p = 0.45). For comparison, the data shown above include only those cells in which the spot and windmill intensities were 8.4 log quanta, but similar results were obtained in other cells from both tiger salamander and mud puppy using windmill stimuli of other intensities.

Fig. 4.

Effect of strychnine and TTX on the waveform of the response elicited by center illumination. The traces show superimposed responses of a tiger salamander on–off ganglion cell to a 400-μm-diameter spot (8.4 log quanta) in the receptive field center in control Ringer’s solution, in the presence of 2 μmstrychnine (STR), and in the presence of 500 nm TTX. Each trace is the average of eight responses. The retina was washed with control Ringer’s solution for 20 min between the two drug applications. Resting potential was −55 mV in all traces.

Fig. 5.

TLI is associated with an increase in ganglion cell conductance. Each trace shows voltage deflections produced in a tiger salamander ganglion cell by a −0.1 nA current pulse (250 msec duration) in the dark (left) and in the presence of a 1200 μm i.d. windmill that was stationary (middle) or rotating (right). Each trace is the average of 20 responses to the current pulse. The voltage deflection produced by the current pulse in darkness was balanced using the bridge circuit of the amplifier, so that a positive voltage deflection indicates an increase in conductance. The downward shift of the right traceindicates the amount of sustained hyperpolarization (−7 mV) produced by the rotating windmill. Resting potential was −58 mV.

Fig. 6.

Effects of strychnine and TTX on light-evoked inhibitory currents in ganglion cells. Responses are from an on–off ganglion cell in a tiger salamander slice preparation. The control Ringer’s solution contained 150 μm picrotoxin to block GABA-mediated responses. The cell was voltage-clamped at 0 mV to eliminate glutamate-mediated excitatory currents. Horizontal bars below responses indicate duration of diffuse light stimulus. A, Currents recorded in control solution (control), in the presence of 2 μmstrychnine (STR), and 20 min after return to control solution (wash). B, Currents recorded in control solution (control), in the presence of 500 nm TTX, and 10 min after return to control solution (wash). All responses were from the same cell. Holding current was 20 pA in A and 34 pA in B. Calibration bars are for both A andB.

Fig. 7.

Effect of strychnine and TTX on glycinergic inhibitory currents elicited by electrical stimulation. IPSCs were recorded from ganglion cells held at 0 mV to eliminate excitatory currents, and the control Ringer’s solution contained 150 μm picrotoxin to block GABA-mediated responses. Responses were elicited by 1 msec, 1 μA current pulses applied through a pipette located in the OPL directly over the recording site (A) and ∼300 μm lateral to the recording site (B). Superimposed traces show current recorded under control conditions (control), in the presence of 2 μm strychnine (STR), and in the presence of 500 nm TTX. A andB were from different cells. Holding current was 15 pA in A and 12 pA in B. Thedot below traces indicates time of electrical stimulation.

Fig. 8.

Effect of strychnine and TTX on responses to direct application of glycine. The superimposed traces show IPSCs recorded from the same ganglion cell in control solution (control), in the presence of 2 μmstrychnine (STR), and in the presence of 500 nm TTX. The cell was voltage-clamped at 0 mV to eliminate excitatory currents, and the control solution contained 150 μm picrotoxin to block GABA-mediated responses. Responses were elicited by a 5 msec puff of 0.5 mm glycine from a pipette located in the inner plexiform layer just above the ganglion cell. The dot below traces indicates time of glycine puff. Holding current was 35 pA.

Fig. 9.

Effect of strychnine and TTX on excitatory input to ganglion cells. The superimposed traces show the inward currents elicited by the same diffuse light flash (indicated byhorizontal line above responses) in control solution (control) and in the presence of 2 μm strychnine (STR) and 500 nmTTX. Strychnine was applied after recovery following washout of TTX. Bath contained 150 μm picrotoxin. Cells were voltage-clamped at the reversal potential for chloride ions (−65 mV) to eliminate glycine-mediated responses. Holding current was −12 pA.

Fig. 10.

Proposed pathway for TLI in mud puppy and salamander retina. Filled and open terminals indicate excitatory and inhibitory synapses, respectively. Glycinergic wide-field transient amacrine cells (GLY WFTA) receive excitatory input from bipolar cells (BC) over a restricted region near their somas and send action potentials laterally (arrows) over long processes (thin lines). Output synapses are onto bipolar terminals and ganglion cell dendrites. It is not known whether GLY–WFTA cells also make feedback synapses onto the bipolar terminals from which they receive excitatory input. Depolarization produced by excitatory inputs can spread passively (over region indicated by thick processes) to affect glycine release at nearby sites, but action potentials are required to elicit glycine release at distant sites (region indicated by thin processes). Alternatively, some or all of the short-range TTX-resistant transient glycinergic inputs to bipolar and ganglion cells may come from a separate population of glycinergic narrow-field transient amacrine cells (not shown). In addition to their role in mediating TLI, glycinergic amacrine cells may also make synapses onto GABAergic amacrine cells (not shown).