**5.2.** *In vitro* **retinal recording and stimulation**

Parylene C-based arrays of thin-film platinum electrodes, comprising four 200 µm diameter stimulating electrodes and 56 recording electrodes of 10 µm diameter were fabricated accord‐ ing to the single-metal-layer process on a glass substrate. These were placed in a bicarbonate perfusate under a microscope and connected to a stimulus generator and preamplification board (Multi Channel Systems MCS GmbH, Reutlingen, Germany) [45]. As shown in Figure 8, a retina isolated from larval tiger salamander (*Ambystoma tigrinum*) was placed RGC side down on the array (to simulate epiretinal stimulation), and a remote platinum ground electrode was introduced to the bath.

**Figure 8.** Isolated larval tiger salamander retina (darker region at left) overlying parylene-based platinum electrode array. Arrow indicates 10 µm diameter electrode used for recording trace in Figure 9. Asterisk identifies 200 µm diam‐ eter stimulating electrode used to generate action potentials seen in Figure 9.

With the lights off, a 20 µA, 400 µs/phase, cathodic-first biphasic electrical pulse was applied between the stimulating electrode indicated with an asterisk in Figure 8 and the ground electrode. The voltage trace from the recording electrode is shown in Figure 9. This stimulation was consistently repeatable over a 50 pulse train with a 400 ms inter-pulse interval, and other stimulating electrodes were also capable of "epiretinally" stimulating other cells in the retinal slice. As is clear from these results, the parylene-based platinum electrode was able to stimulate the tissue and elicit a response similar to the response generated from a light pulse in this intact retina. Given these results and the knowledge garnered from clinical trials with prototype arrays fabricated of other materials, it is not unreasonable to presume that our arrays will most likely be able to stimulate retinal tissue in other species, including human.

**Figure 9.** Typical recording of response of cells overlying recording electrode to a 20 µA, 400 µs/phase, cathodic-first biphasic electrical pulse from "epiretinal" stimulating parylene-based platinum electrode denoted with an asterisk in Figure 8.
