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.

Light-Evoked Modulation of Neuronal Activity with Conjugated Polymers

Diego Ghezzi, Mattia Nova

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.

2015

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

Optogenetic Probing of Cortical Synaptic Circuits Measured Through in Vivo Whole-Cell Recordings in the Mouse Barrel Cortex

Céline Matéo

Although neocortex underlies higher-order brain functions, little is known about the synaptic interactions that drive neocortical microcircuit function in vivo. The neocortex is spontaneously active in vivo so I explored how such spontaneous network activity affects in vivo the interactions between excitatory and inhibitory neurons in the barrel cortex. To this aim, I have focused on the layer 2/3 microcircuit of the C2 barrel column driving sensory processing for the C2 whisker, and combined in vivo whole-cell patch-clamp recordings in anesthetised and awake head-restrained mice with optogenetic stimulation, that allows controlling the activity of genetically defined neurons with light. I expressed channelrhodopsin-2 (ChR2), a light-gated cation channel to evoke action potentials in excitatory neurons with brief light pulses. The impact of this optogenetic stimulus was measured on the membrane potential dynamics of their postsynaptic target neurons. The optogenetic stimulus drove reliable action potentials in ChR2-expressing excitatory neurons, independent of the state of ongoing spontaneous cortical activity. In contrast, I found a marked modulation of light-evoked postsynaptic responses in ChR2-non-expressing excitatory neurons by cortical activity. During spontaneously hyperpolarised periods (DOWN state), optogenetic stimulation resulted in depolarisations of postsynaptic excitatory neurons, while during depolarised periods (UP state), the response was significantly smaller and shorter. The optogenetic stimulus drove the membrane potential of postsynaptic excitatory layer 2/3 neurons towards reversal potentials, which were hyperpolarised relative to action potential threshold, leading to a reduction in firing rates. Targeting whole-cell recordings to GFP-labelled GABAergic layer 2/3 neurons with two-photon microscopy revealed that both fast-spiking and non-fast-spiking inhibitory postsynaptic neurons were rapidly driven to fire action potentials, preferentially from UP states. Such rapid activation of inhibitory neurons in the UP state is likely to be responsible for decreasing firing rate in excitatory neurons. As a result, optogenetic stimulation of a given set of excitatory neurons results in a state-dependent recruitment of inhibitory neurons that limit the spread of excitation within the layer 2/3 microcircuit. The data presented here indicates that a powerful inhibitory loop within the cortical microcircuit rapidly balances excitation with inhibition and enforces sparse coding of layer 2/3 excitatory neurons.

EPFL2011