We used cell-attached patch clamp recordings to measure the spiking response of retinal ganglion cells to electric stimulation. We found that a relatively tight seal (~40
MΩ) allowed large amplitude spikes to be recorded without obstruction from the stimulus artifact. Typical responses to sinusoidal stimulation and to stimulation with white noise are shown in Figures A,B, respectively. Robust spiking was observed at all sinusoidal stimulus frequencies tested (5–100
Hz), however cells were most sensitive to low frequencies. In fact, some cells responded to 5–10
Hz stimulation at the lowest stimulus levels we could deliver (1
Figure 1 Spike recordings using cell-attached patch clamping allows visualization of the spikes (*) through the stimulus artifact, as shown for sinusoidal (A) and binary white noise (B) stimulation. Both stimulus waveforms elicited robust spiking responses. The (more ...)
Electric stimulation with white noise was highly effective at activating some cells, while other cells did not elicit a response even at the highest stimulus amplitude tested (peak-to-peak 4.4
μA). Following stimulation for 60
s with white noise, the spike times were extracted and the spike-triggered average (STA) was computed by averaging the stimulus waveform that preceded each spike (Rieke et al., 1997
). The STA represents the best linear description of the neuronal response to electric stimulation. A typical STA from an ON-ganglion cell is shown in Figure C. The STA has two peaks (Figure C): an early peak (<5
ms) and a late phase peak (~25
ms). The late phase and overshoot of the STA were abolished following application of the synaptic blocker cadmium chloride (not shown), suggesting that the early peak of the STA is mediated by direct excitation, while the late phase is mediated by activation of presynaptic neurons.
Retinal ganglion cells receive synaptic inputs from excitatory bipolar cells and inhibitory amacrine cells. By voltage clamping the ganglion cell membrane potential at −60
mV, we isolated the excitatory currents in response to electric stimulation. Examples are shown for stimulation with sinusoidal stimulation (Figure D) and stimulation with noise (Figure E). By convention, inward, excitatory currents are depicted as negative, downward deflections. Consistent with the spiking response, both sinusoidal and white noise stimulation elicited robust excitatory currents in the ganglion cell.
White noise analysis was used to estimate the temporal relationship between the electric stimulus and the resulting current. The stimulus artifact is very small compared to the excitatory currents, as seen in Figure E where the rapid fluctuations in the response (~50
pA peak-to-peak) do not obstruct the input currents, which are hundreds of picoamps. After collecting 60
s of white noise data, the raw current response was cross-correlated with the stimulus waveform, giving an estimate of the linear kernel (Figure F). As with the STA in Figure C, the peak of the kernel occurs at about 25
ms, followed by an overshoot. The early peak (<1
ms) of the kernel likely results from a capacitive transient, which is present for whole-cell recordings since the cell membrane now separates the stimulating electrode from the recording electrode.