at 10 Hz. These findings suggest that temporal frequencies may be limited to <5 Hz if bipolar cells are activated (from either subor epiretinal stimulation).

Since activation of bipolar cells leads to activation of amacrine cells [28], the reduction in bipolar cell activity may arise from amacrine cell mediated feedback inhibition. Therefore, methods that reduce or eliminate the secondary activation of amacrine cells are likely to enhance the temporal response to stimulation. Such methods have yet to be developed.

12.2.2  Subretinal Stimulation

12.2.2.1  Target of Stimulation

Similar to epiretinal stimulation, subretinal stimulation activates many different classes of retinal neurons. Stett et al. [53] used pharmacological blockade of synaptic pathways to explore which classes of (chicken) retinal neurons were activated by subretinal stimulation. Under control conditions, 0.5 ms monophasic voltage pulses elicited RGC spiking responses whose durations lasted up to several hundred milliseconds (Fig. 12.10). Addition of magnesium (Mg2+), a general blocker of neurotransmitter release, significantly reduced the RGC responses. The Mg2+ sensitive spiking activity in RGCs presumably results from activation of one or more presynaptic excitatory neurons; the likely candidates are photoreceptors, bipolar cells and/or starburst amacrine cells. To identify the specific neurons that were activated, synaptic blockers of the excitatory neurotransmitter glutamate were administered. Application of kynurenic acid, an AMPA/kainate receptor antagonist, greatly reduced the RGC responses to electrical stimulation. Kynurenic acid targets receptors at multiple locations [14, 57] but notably it blocks the output of bipolar cells. A more specific glutamate receptor blocker, 2-amino-4-phosphonobutyric acid (AP4), blocks the synapse between photoreceptors and ON bipolar cells [51]. Application of AP4 abolished the electrically evoked responses. Although Stett et al. [53] did not identify the physiological type of RGC shown in Fig. 12.10, the fact that AP4 abolished the evoked response suggests that this RGC cell was an ON cell. Jensen et al. [24] reported in a later study (using epiretinal stimulation) that electrically evoked responses of ON RGCs but not OFF RGCs in rabbit retina were abolished with AP4.

The AP4 results suggest that photoreceptors are the principal target of electrical stimulation in normal retina. Understanding whether photoreceptors or bipolar cells are the target of subretinal stimulation has important implications for clinical use since patients that have been blind for many years will have few or no viable photoreceptors remaining (Chap. 3). Therefore methods that target photoreceptors are not likely to be useful in clinical applications. More research is needed to determine the relative excitability between photoreceptors and bipolar cells.

Fig. 12.10  Synaptic blockers reduce the response to subretinal stimulation. Response histograms (5 ms bin width) elicited by 20 repetitions of a single voltage pulses (2 V, 0.5 ms). Application of high [Mg2+], kynurenic acid or AP4 reduced the spiking activity. The number at each histogram indicates the time interval (minutes) after switching to the perfusate with the agents given at the right and to the standard perfusate for washing out the agents. Scale bars 100 ms, 100 spikes/s. Reprinted from [53], Fig. 6, with permission

12.2.2.2  Threshold vs. Polarity of Stimulation Pulse

It is well known that axons (including those of RGCs) are more sensitive to a cathodal current pulse than to an anodal current pulse [56]. When RGCs are activated through electrical stimulation of presynaptic cells, the situation is not so straightforward.

Jensen and Rizzo [19] reported that when the rabbit retina is stimulated with a subretinal electrode the threshold current needed to activate OFF RGCs was much lower for an anodal current pulse than for a cathodal current pulse. On the other hand, cathodal and anodal current pulses were on average equally effective for activating ON RGC cells. This is illustrated in Fig. 12.11, in which threshold measurements were made for RGC responses to stimulation of the neural network.

Fig. 12.11  Threshold charge as a function of stimulus pulse duration for OFF ganglion cells (left graph) and ON ganglion cells (right graph) for both cathodal and anodal stimulus pulses. For OFF ganglion cells (left), anodal stimulation produces a substantially lower activation threshold for all pulse durations, while as a whole ON ganglion cells (right) are insensitive to the polarity of stimulation. Reprinted from [19], Figs. 4 and 5, with permission

In the chicken retina, Stett et al. [54] reported that when the neural network is stimulated with a subretinal electrode, anodal voltage pulses were overall more effective than cathodal voltage pulses. They found that on average a 3.2-fold difference in thresholds. They did not distinguish between ON and OFF RGCs. Nevertheless, both studies suggest that for indirect activation of RGCs (with a subretinal electrode) an anodal stimulus is in general more effective than a cathodal stimulus. The findings of Jensen and Rizzo [19] further suggest that a cathodal current pulse may bias activation of ON cells over OFF cells. Results such as these may one day underlie methods to selectively activate ON vs. OFF pathways which would allow more physiological patterns of activity to be elicited.

In contrast to the ON-OFF selectivity found in rabbit (described above), a recent study conducted in the mouse retina [22] found that the median threshold current for cathodal stimulation of ON RGCs was only 32% lower than for OFF RGCs and this difference was not statistically significant. Thus, it would seem from the mouse experiments that a cathodal current pulse may not bias activation of ON cells over OFF cells as the findings in the rabbit would suggest. It will be of interest to examine the thresholds of ON and OFF RGCs to anodal and cathodal current pulses in the primate retina.

12.2.2.3  Spatial Extent of Activation

Stett et al. [54] examined the spatial extent of activation of RGCs in the chicken retina. They used an ultra-fine (1-mm diameter) tip electrode for stimulating the retina and a dense multielectrode array to record simultaneously from many RGCs in the retina.

They reported a half width of an “electrical point spread function” of ~100 mm. This distance on the retina corresponds to a visual angle of 21¢ in the human eye. A minimum angle of resolution of 21¢ corresponds to a visual acuity of ~20/400. It will be of interest to examine the electrical point spread function of RGCs in the primate fovea where the convergence of photoreceptors and bipolar cells onto RGCs is very low. The findings may indicate that a higher visual acuity is possible.

12.2.2.4  Temporal Response Properties

Fried et al. [8] showed that when rabbit RGCs are indirectly activated with an epiretinal stimulating electrode, bipolar cell output is drastically reduced by a 10 Hz stimulation frequency. The situation is not much different with a subretinal stimulating­ electrode. Jensen and Rizzo [20] showed that the responses of rabbit RGCs to stimulation of the neural network began to diminish in size when the retina was stimulated within ~400 ms of a preceding current pulse (Fig. 12.12). The shorter the interpulse interval, the smaller was the response to the second stimulation pulse. They also studied the responses of RGCs to trains of pulses applied at different frequencies­ . As expected, the responses were greatly reduced for stimulation frequencies­ >25 Hz. These data indicate that rapid electrical stimulation of the retina in patients with a retinal prosthesis may be counterproductive, assuming that RGCs are being activated through the neural network.

Fig. 12.12  Mean pairedpulse depression of RGC cell response amplitudes in rabbit retina. Data were collected using biphasic current pulses of 1 ms per phase. Amp1 amplitude of first response; Amp2 amplitude of second response. Reprinted from [20], Fig. 2, with permission