Light-Evoked Modulation of Neuronal Activity with Conjugated Polymers

Organic bioelectronics aims at the intimate integration between organic materials and biological tissues in order to obtain a targeted functional outcome. In the last decades, conductive polymers have been successfully employed to interface excitable cells through biocompatible probes and devices. We previously reported the possibility to culture primary neurons on photosensitive conjugated polymers, such as poly(3-hexylthiophene) (P3HT), without altering either the polymer or the neuronal properties. These neurons displayed a depolarizing response to a brief (< 50 ms) green light stimulus and a consequent control over the action potential firing rate. The same interface was also shown to elicit activation of retinal circuitry and ganglion cell firing in explanted degenerate retinas upon light stimulation. However, while neuronal excitation has been extensively proved via planar or protruding electrodes, carbon nanotubes, and conjugated polymers, the hyperpolarization of neurons and silencing of their firing activity has been achieved with optogenetics, but rarely documented for functionalized planar devices and structures. We show here a method to induce both depolarization and hyperpolarization of cells interfaced with conjugated polymers upon prolonged (500 ms) light stimulation. Patch-clamp experiments showed that HEK293 cells grown on P3HT displayed a biphasic response composed of an initial depolarization followed by a sustained hyperpolarization during light illumination. We also documented that the amplitude of the light-induced hyperpolarization was reduced in the absence of intracellular potassium, but was insensitive to changes in the intracellular chloride concentration. A prolonged illumination was equally able to hyperpolarize primary neurons cultured over P3HT and significantly reduced both spontaneous and electrically elicited action potential frequency. We further assessed the translatability of our findings to tissue stimulation by describing the modulation of ganglion cell firing in explanted blind retinas from Royal College of Surgeons rats, a model of Retinitis pigmentosa. By coupling conjugated polymers with multi-electrode arrays and conventional metal electrodes, single-unit activity of ganglion cells was inhibited by prolonged illumination. We similarly investigated the phenomenon in acute hippocampal brain slices placed onto a conjugated polymer thin film. These results indicate that conjugated polymers can be used to non-invasively excite and inhibit the electrical activity of cells cultured on its surface. Furthermore, the translation of our findings from in vitro cultures to different central nervous system tissues, suggests a potential application of these organic devices as a tool for modulation of neuronal activity in vivo

Hemichannel-Mediated and pH-Based Feedback from Horizontal Cells to Cones in the Vertebrate Retina

Background: Recent studies designed to identify the mechanism by which retinal horizontal cells communicate with cones have implicated two processes. According to one account, horizontal cell hyperpolarization induces an increase in pH withinthe synaptic cleft that activates the calcium current (Ca2+-current) in cones, enhancing transmitter release. An alternative account suggests that horizontal cell hyperpolarization increases the Ca2+-current to promote transmitter release through ahemichannel-mediated ephaptic mechanism.Methodology/Principal Findings: To distinguish between these mechanisms, we interfered with the pH regulating systems in the retina and studied the effects on the feedback responses of cones and horizontal cells. We found that the pH buffers HEPES and Tris partially inhibit feedback responses in cones and horizontal cells and lead to intracellular acidification ofneurons. Application of 25 mM acetate, which does not change the extracellular pH buffer capacity, does lead to both intracellular acidification and inhibition of feedback. Because intracellular acidification is known to inhibit hemichannels, the key experiment used to test the pH hypothesis, i.e. increasing the extracellular pH buffer capacity, does not discriminatebetween a pH-based feedback system and a hemichannel-mediated feedback system. To test the pH hypothesis in a manner independent of artificial pH-buffer systems, we studied the effect of interfering with the endogenous pH buffer, the bicarbonate/carbonic anhydrase system. Inhibition of carbonic anhydrase allowed for large changes in pH in the synapticcleft of bipolar cell terminals and cone terminals, but the predicted enhancement of the cone feedback responses, according to the pH-hypothesis, was not observed. These experiments thus failed to support a proton mediated feedback mechanism. The alternative hypothesis, the hemichannel-mediated ephaptic feedback mechanism, was therefore studied experimentally, and its feasibility was buttressed by means of a quantitative computer model of the cone/horizontal cellsynapse.Conclusion: We conclude that the data presented in this paper offers further support for physiologically relevant ephaptic interactions in the retina

Parameters of photopigment template fits.

<p>Mean parameter values for the various cone types. Parameters are presented as mean ± <i>SD.</i></p

Fits of pigment template to experimental data.

<p>This figure displays the fits (solid lines) of the photopigment template <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068540#pone.0068540-Govardovskii1" target="_blank">[15]</a> to the average experimental data per cone-type with the peak wavelength of the A1-based photopigment, the ratio between A1- and A2-based photopigment and the presence of the β-wave relative to the original photopigment template as free parameters. For comparison the spectral sensitivity functions of corresponding A1- (dashed lines) and A2-based (dotted lines) photopigments are also plotted. These were constructed according to the generic photopigment template in combination with their peak absorbance wavelength as measured <i>in vitro </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068540#pone.0068540-Chinen1" target="_blank">[7]</a>. The UV-cone action spectra deviates considerable from the UV template, presumably due to the small number of data points (top row, left). The action spectrum of the SWS cones was fully overlapped the SWS-2 adsorption spectrum (top row, right). For the MWS cones, both RH2–2 and RH2–3 templates were covering the MWS action spectrum, while RH2–1 and RH2–4 templates could not describe the action spectrum properly. Finally both LWS-1 and LWS-2 templates covered the LWS action spectrum.</p

Properties of zebrafish cones.

<p>Average properties of the various zebrafish cones types. V_rest, resting membrane potential; R_max, maximum response amplitude relative to V_rest; n, coefficient of fit Hill-relation; S_abs absolute sensitivity (see Methods section for details). Parameters are presented as mean ± <i>SD.</i></p

Action spectra of individual cells and averages per cone-type.

<p>In the top four panels the relative sensitivity is plotted against the stimulus wavelength of individual cones grouped according to cone-type as indicated in the graph. The bottom panel displays the average spectral sensitivity for the different cone-types.</p

Comparison of zebrafish cone spectral sensitivity data.

<p>λ_max values (in nm) of zebrafish A1-based photopigments and cone-types from literature. Parameters are presented as mean ± <i>SD</i>. <i>^a</i> Chinen et al. (2003); <i>^b</i> Nawrocki et al. (1985); <i>^c</i> Robinson et al. (1993); <i>^d</i> Cameron (2002); <i>^e</i> Govardovskii et al. (2000); <i>^f</i> Allison et al. (2004).</p

Light-evoked hyperpolarization and silencing of neurons by conjugated polymers

The ability to control and modulate the action potential firing in neurons represents a powerful tool for neuroscience research and clinical applications. While neuronal excitation has been achieved with many tools, including electrical and optical stimulation, hyperpolarization and neuronal inhibition are typically obtained through patch-clamp or optogenetic manipulations. Here we report the use of conjugated polymer films interfaced with neurons for inducing a light-mediated inhibition of their electrical activity. We show that prolonged illumination of the interface triggers a sustained hyperpolarization of the neuronal membrane that significantly reduces both spontaneous and evoked action potential firing. We demonstrate that the polymeric interface can be activated by either visible or infrared light and is capable of modulating neuronal activity in brain slices and explanted retinas. These findings prove the ability of conjugated polymers to tune neuronal firing and suggest their potential application for the in-vivo modulation of neuronal activity