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.

A polymer-based interface restores light sensitivity in blind rats

Diego Ghezzi

Progressive degeneration of photoreceptors is one of the major causes of adult blindness in “industrialized” countries; as an example, Retinitis pigmentosa defines a set of monogenic hereditary retinal diseases, with a prevalence of 1:4000 worldwide, caused by single mutations in over 50 genes. Moreover, it is thought that a significant number of implicated genes still await identification (Hartong et al., 2006;Wright et al., 2010;Daiger et al., 2013). In the past 20 years, several treatments have been attempted to treat Retinitis pigmentosa as well as many other retinal genetic diseases, including: drug therapy ( Leveillard and Sahel, 2010), gene therapy (Smith et al., 2012), stem/progenitor cells engrafting (Pearson et al., 2012), optogenetic manipulation (Busskamp et al., 2010), and implantation of prostheses (Weiland et al., 2011;Zrenner et al., 2011). However, despite the enormous efforts and advances in the clinical treatment of eye diseases and some encouraging results in animal models, there is currently no effective cure for the majority of genetic retinal diseases affecting photoreceptors including Retinitis pigmentosa. Sight restoration represents one of the new frontiers for prosthetic devices enabling the electrical stimulation of neurons. In particular, diseases that affect the retinal pigment epithelium and photoreceptors but preserve the inner retinal layers are preferential targets for the implantation of visual prostheses. In our work, we exploited the use of conjugated polymers to generate an organic photovoltaic retinal prosthesis. Our research indicates that organic materials, in particular photovoltaic semiconducting polymers, are suitable for the generation of a fully organic retinal prosthesis, to restore light sensitivity in blindness caused by photoreceptor degeneration. We discovered that primary neurons can be grown onto a semiconductor polymer layer, without affecting the optoelectronic properties of the photovoltaic material (Antognazza et al., 2009) or the physiological properties of the neuronal network over a long period of time. Moreover, we found that a light pulse is able to depolarize neurons and induce them to fire action potentials (Ghezzi et al., 2011). The spatial resolution of the stimulation paradigm was demonstrated to be in the order of the cell body, in spite of the continuous layer of polymer, indicating that the stimulation is strictly confined to the illuminated area. This result encouraged us to test the efficacy of photovoltaic polymers in stimulating retinas explanted from albino rats with reproducibly light-induced degeneration of the photoreceptor layer. To this aim, acutely dissected retinas were placed on the organic polymer in a sub-retinal configuration (i.e., external layers in contact with the polymer). Light stimulation of the degenerate retina was observed by monitoring multi-unit activity and field potentials with an extracellular electrode positioned in the retinal ganglion cell layer. Multi-unit activity recordings showed that a light stimulus 16-fold lower than the safe limit for pulsed illumination elicited intense spiking activity in degenerate retinas placed on polymer-coated substrates to levels indistinguishable from those recorded in control retinas. Moreover, to evaluate the efficiency of the interface, a dose-response analysis of spiking activity versus light intensity were performed in degenerate retinas. Spiking activity was observed in degenerate retinas over the polymer with a response threshold below 0.3 μW/mm2, a linear increase in a range corresponding to daylight irradiance, and a response saturation above 100 μW/mm2 (considered the safe limit for chronic illumination). A 4-fold increase in the amplitude of the light response at saturation and a significant left shift of the dose-response curves were obtained in retinas placed over the polymer-coated interface respect to degenerate retinas on glass substrates. Our finding indicates that the interface could mimic functional photoreceptors in activating the processing of the inner retina and is able to rescue normal light sensitivity (Ghezzi et al., 2013). Following these promising results, we implanted the prosthesis in the eye of rats (Royal College of Surgeons) bearing photoreceptor degeneration due to mutation in the MERTK gene (D'Cruz et al., 2000). These rats are a recognized animal model of human Retinitis pigmentosa. Preliminary experiments performed in a first cohort of animals show that the retina remains well attached over the entire region of the sub- retinal implant without inflammation or fibrosis. Moreover, the ability of the organic prosthesis to restore light sensitivity was first tested by monitoring the pupillary reflex. Pupil constriction in the implanted blind rat was similar to the response of the non-dystrophic animal and significantly higher than the non-implanted blind animal. Finally, visually evoked field potentials were detected in the visual cortex of implanted blind rats, whereas they were undetectable in the non-implanted blind animals. These results open the possibility of developing a new generation of fully organic prosthetic devices for sub-retinal implantation.

2014

Light-evoked hyperpolarization and silencing of neurons by conjugated polymers

Diego Ghezzi, Mattia Nova

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.

2016

Impact of Carbon-Nanotube Substrate Coating in Neuronal Networks

Luca Gambazzi

The interest in new materials and their application in the neuroscience are growing incredibly fast. Current research interests are increasingly focusing on new artificial materials and their direct effects on the central nervous systems so that it would be possible use them to restore the original nervous function lost due to neuronal diseases or trauma. This thesis is part of the international Neuronano project and has been funded by the European Commission 6th Framework Programme. The main goal is to understand the interactions of carbon nanotubes (CNT) with the nervous tissues. Not only it focuses on how neuron stimulations and recordings instruments may be ameliorated by a CNT-neuron interface, but it also focuses on the endogenous effects of those artificial materials on neuronal activity. This thesis is divided into four themes: First, an integrated CNT-neuron system was developed to test whether electrical voltage stimulation, delivered via nanotubes, can induce neuronal signaling. It was also compared to neuronal responses evoked by voltage steps delivered via conventional patch-pipette. In the second part, the focus was on understanding the CNT-neuron interface and the tight contacts with the cell membranes that might favour electrical shortcuts between neuronal compartments. The third part is an overview of the effects of a CNT coated substrates when employed to grow long-term cell cultures. The activity was constantly monitored by an extracellular array of electrodes (MEA) for several weeks. The intimate relationship between nanotubes and cell membrane observed at single-neuron and network level, results in a more robust network activity. Finally, the MEA setup developed for cell cultures monitoring was used to investigate the effects of of BDNF on cortical cell culture model of Huntington disease.

EPFL2010

A polymer-based interface restores light sensitivity in blind rats

Diego Ghezzi

2014