A polymer optoelectronic interface restores light sensitivity in blind rat retinas

Abstract

Interfacing organic electronics with biological substrates offers new possibilities for biotechnology due to the beneficial properties exhibited by organic conducting polymers. These polymers have been used for cellular interfaces in several fashions, including cellular scaffolds, neural probes, biosensors and actuators for drug release. Recently, an organic photovoltaic blend has been exploited for neuronal stimulation via a photo-excitation process. Here, we document the use of a single-component organic film of poly(3-hexylthiophene) (P3HT) to trigger neuronal firing upon illumination. Moreover, we demonstrate that this bio-organic interface restored light sensitivity in explants of rat retinas with light-induced photoreceptor degeneration. These findings suggest that all-organic devices may play an important future role in sub-retinal prosthetic implants.

Figure 1. Characterisation of the photo-stimulus generated by the polymeric interface

(a) Schematic representation of the stimulation and recording paradigm. A patch-clamp amplifier was used to detect photo-currents generated upon light stimulation through a patch pipette positioned in close proximity (<5 μm) to the P3HT surface. (b) Photo-current detected in voltage-clamp mode upon light illumination (20 ms, 15 mW/mm²; green bar). The trace represents an average of 5 consecutive sweeps. (c) Photo-currents generated upon repetitive light pulses (20 ms, 15 mW/mm²; green bars) at a repetition rate of 2 Hz. A substantial preservation of the photo-current during the light pulse train was observed. (d) Distribution of the photo-current along the P3HT surface and at increasing distances from the polymer surface. The green circle represents the light spot (100 μm, 20 ms, 15 mW/mm²), whereas the white and red dots represent the points at which the patch pipette was sequentially positioned. The black dot represents the starting position. (e) The mean (± s.e.m.) photo-current along the P3HT surface, normalised to the amplitude of the first response, is shown as a function of distance from the spot centre (n = 6). (f) Photo-current detected in voltage-clamp mode upon light illumination at increasing distances from the polymer surface. The right panel shows the mean (± s.e.m.) photo-current intensity, normalised with respect to the amplitude of the first response, as a function of distance from the polymer surface (n = 6).

Figure 2. Photovoltaic excitation of neurons mediated by a P3HT active layer

(a) Schematic representation of the stimulation and recording paradigm. Neuronal responses to light illumination (20 ms, 15 mW/mm²) were detected by patch clamp in current-clamp mode. (b) Activation by a light pulse (green bar) of a representative neuron cultured on a P3HT-coated Glass:ITO substrate (black) with respect to a neuron cultured on a control Glass:ITO coverslip (grey). The right plot shows the mean (± s.e.m.) latency to the spike peak with respect to light onset and latency jitter calculated as the standard deviation of spike latencies measured across all recorded neurons (n = 21). (c) Neuronal activation at various frequencies with a train of 20 stimuli (indicated by the green bars). The inset (red square) shows the spike or failure responses to the 20 Hz stimulation. (d) The percentage of successful spikes in the train of 20 pulses was computed over all recorded neurons and reported as a function of the stimulation frequency (n = 12, means ± s.e.m.).

Figure 3. The photoreceptor layer is replaced in the degenerate retina by the organic polymer

(a) Schematic illustrations of the retinal structure (left) and the stimulation/recording interface for degenerate retinas (right). (b) Confocal images of latero-dorsal control (left) and degenerate (right) retinal sections labelled with the nuclear stain bisbenzimide (gcl: ganglion cell layer; inl: inner nuclear layer; onl: outer nuclear layer). Scale bar, 50 μm.

Figure 4. The P3HT layer restores responses in blind retinas

(a) Top panels show MUAs recorded upon light stimulation (10 ms, 4 mW/mm²) of a control retina over a Glass:ITO substrate (left), a degenerate retina over a Glass:ITO substrate (middle) and a degenerate retina over a P3HT-coated Glass:ITO substrate (right). The bottom panels show normalised post-stimulus time histograms (PSTHs, bin: 25 ms) computed based on all sweeps recorded in single retinas (10 ms, 4 mW/mm²) for the three experimental conditions. Green bars/arrows represent the light stimulus. (b) Comparison of mean (± s.e.m.) PSTHs (bin: 25 ms) obtained from control retinas on Glass:ITO (black bars, n = 5), degenerate retinas on Glass:ITO (open bars, n = 10) and degenerate retinas on P3HT-coated Glass:ITO (red bars, n = 10) in response to light illumination (10 ms, 4 mW/mm²; green arrow). Significantly different bins are indicated (Student’s t-test, P < 0.001). (c) Dose-response analysis of the mean (± s.e.m.) firing rate versus light intensity performed in degenerate retinas over P3HT-coated Glass:ITO (red dots, n = 6) or Glass:ITO alone (open dots, n = 6). Mean firing rates were calculated in a window of 250 ms after the light pulse. The dashed line represents the computed maximum permissible radiant power for a chronic exposure (see Methods). Dose-response curves were fitted using a sigmoidal dose-response model. Solid grey lines represent the response threshold (10% of the maximal response), and dotted grey lines represent the average ED₅₀ calculated from the fitting procedure (12.11 and 120.78 μW/mm², respectively). On the right, representative PSTHs (bin: 25 ms, means ± s.e.m.) obtained in the presence (red) or absence (grey) of P3HT are shown. The green arrow represents the light stimulus.