Konstantin Nikolic

Multi-site optical excitation using ChR2 and micro-LED array.

Studying neuronal processes such as synaptic summation, dendritic physiology and neural network dynamics requires complex spatiotemporal control over neuronal activities. The recent development of neural photosensitization tools, such as channelrhodopsin-2 (ChR2), offers new opportunities for non-invasive, flexible and cell-specific neuronal stimulation. Previously, complex spatiotemporal control of photosensitized neurons has been limited by the lack of appropriate optical devices which can provide 2D stimulation with sufficient irradiance. Here we present a simple and powerful solution that is based on an array of high-power micro light-emitting diodes (micro-LEDs) that can generate arbitrary optical excitation patterns on a neuronal sample with micrometre and millisecond resolution. We first describe the design and fabrication of the system and characterize its capabilities. We then demonstrate its capacity to elicit precise electrophysiological responses in cultured and slice neurons expressing ChR2.

Photocycles of Channelrhodopsin-2

Recent developments have used light‐activated channels or transporters to modulate neuronal activity. One such genetically‐encoded modulator of activity, channelrhodopsin‐2 (ChR2), depolarizes neurons in response to blue light. In this work, we first conducted electrophysiological studies of the photokinetics of hippocampal cells expressing ChR2, for various light stimulations. These and other experimental results were then used for systematic investigation of the previously proposed three‐state and four‐state models of the ChR2 photocycle. We show the limitations of the previously suggested three‐state models and identify a four‐state model that accurately follows the ChR2 photocurrents. We find that ChR2 currents decay biexponentially, a fact that can be explained by the four‐state model. The model is composed of two closed (C1 and C2) and two open (O1 and O2) states, and our simulation results suggest that they might represent the dark‐adapted (C1‐O1) and light‐adapted (C2‐O2) branches. The crucial insight provided by the analysis of the new model is that it reveals an adaptation mechanism of the ChR2 molecule. Hence very simple organisms expressing ChR2 can use this form of light adaptation.

Optobionic vision?a new genetically enhanced light on retinal prosthesis

The recent discovery that neurons can be photostimulated via genetic incorporation of artificial opsins is creating a revolution in the field of neural stimulation. In this paper we show its potential in the field of retinal prosthesis. We show that we need typically 100 mW cm(-2) in instantaneous light intensity on the neuron in order to stimulate action potentials. We also show how this can be reduced down to safe levels in order to negate thermal and photochromic damage to the eye. We also describe a gallium nitride LED light source which is also able to generate patterns of the required intensity in order to transfer reliable images.

Micro-LED arrays: a tool for two-dimensional neuron stimulation

Stimulating neuron cells with light is an exciting new technology that is revolutionizing the neurosciences. To date, due to the optical complexity that is involved, photostimulation has only been achieved at a single site using high power light sources. Here we present a GaN based micro-light emitting diode (LED) array that can open the way to multi-site photostimulation of neuron cells. The device is a two-dimensional array of micrometre size LED emitters. Each emitter has the required wavelength, optical power and modulation bandwidth to trigger almost any photosensitizer and is individually addressable. We demonstrate micrometre resolution photoactivation of a caged fluorophore and photostimulation of sensitized living neuron cells. In addition, a complete system that combines the micro-LED array with multi-site electrophysiological recording based on microelectrode array technology and/or fluorescence imaging is presented.

Modeling Study of the Light Stimulation of a Neuron Cell With Channelrhodopsin-2 Mutants

Channelrhodopsin-2 (ChR2) has become a widely used tool for stimulating neurons with light. Nevertheless, the underlying dynamics of the ChR2-evoked spikes are still not yet fully understood. Here, we develop a model that describes the response of ChR2-expressing neurons to light stimuli and use the model to explore the light-to-spike process. We show that an optimal stimulation yield is achieved when the optical energies are delivered in short pulses. The model allows us to theoretically examine the effects of using various types of ChR2 mutants. We show that while increasing the lifetime and shuttering speed of ChR2 have limited effect, reducing the threshold irradiance by increased conductance will eliminate adaptation and allow constant dynamic range. The model and the conclusion presented in this study can help to interpret experimental results, design illumination protocols, and seek improvement strategies in the nascent optogenetic field.

A Simulation Study of the Combined Thermoelectric Extracellular Stimulation of the Sciatic Nerve of the Xenopus Laevis: The Localized Transient Heat Block

The electrical behavior of the Xenopus laevis nerve fibers was studied when combined electrical (cuff electrodes) and optical (infrared laser, low power sub-5 mW) stimulations are applied. Assuming that the main effect of the laser irradiation on the nerve tissue is the localized temperature increase, this paper analyzes and gives new insights into the function of the combined thermoelectric stimulation on both excitation and blocking of the nerve action potentials (AP). The calculations involve a finite-element model (COMSOL) to represent the electrical properties of the nerve and cuff. Electric-field distribution along the nerve was computed for the given stimulation current profile and imported into a NEURON model, which was built to simulate the electrical behavior of myelinated nerve fiber under extracellular stimulation. The main result of this study of combined thermoelectric stimulation showed that local temperature increase, for the given electric field, can create a transient block of both the generation and propagation of the APs. Some preliminary experimental data in support of this conclusion are also shown.

Modeling and engineering aspects of channelrhodopsin2 system for neural photostimulation.

It is desirable to be able to stimulate neural cells for many different therapeutic applications. Light stimulation has many advantages over electrical stimulation if it can be achieved. Neural cells are not naturally light sensitive but they can be transformed using different strategies. Here we examine the case of genetically engineered neurons expressing green algae light-gated ion channels, Channelrhodopsin-2. We have developed a mathematical model for the photocycle of this protein, which gives results which are in good agreement with experimental measurements. We have also examined engineering aspects of using this ChR2 system as a phototransduction mechanism. The response characteristics were calculated and potentials of this system-device are discussed

The spatial pattern of light determines the kinetics and modulates backpropagation of optogenetic action potentials

Optogenetics offers an unprecedented ability to spatially target neuronal stimulations. This study investigated via simulation, for the first time, how the spatial pattern of excitation affects the response of channelrhodopsin-2 (ChR2) expressing neurons. First we described a methodology for modeling ChR2 in the NEURON simulation platform. Then, we compared four most commonly considered illumination strategies (somatic, dendritic, axonal and whole cell) in a paradigmatic model of a cortical layer V pyramidal cell. We show that the spatial pattern of illumination has an important impact on the efficiency of stimulation and the kinetics of the spiking output. Whole cell illumination synchronizes the depolarization of the dendritic tree and the soma and evokes spiking characteristics with a distinct pattern including an increased bursting rate and enhanced back propagation of action potentials (bAPs). This type of illumination is the most efficient as a given irradiance threshold was achievable with only 6 % of ChR2 density needed in the case of somatic illumination. Targeting only the axon initial segment requires a high ChR2 density to achieve a given threshold irradiance and a prolonged illumination does not yield sustained spiking. We also show that patterned illumination can be used to modulate the bAPs and hence spatially modulate the direction and amplitude of spike time dependent plasticity protocols. We further found the irradiance threshold to increase in proportion to the demyelination level of an axon, suggesting that measurements of the irradiance threshold (for example relative to the soma) could be used to remotely probe a loss of neural myelin sheath, which is a hallmark of several neurodegenerative diseases.

High-sensitivity silicon retina for robotics and prosthetics

Conventional image sensors differ fundamentally from biological retinas, because they produce redundant image sequences at a limited frame rate. Understanding the principles by which biological eyes achieve their superior performance and implementing them in artificial retinas are scientific and technical challenges with far-reaching implications in the clinical, neuroscience, and technological fields. Despite a more than 100-year effort to understand the retina, the function of most of its cells remains unknown. However, recent breakthroughs in genetics, viral trans-synaptic tracing, and two-photon, laser-targeted electrophysiology (see Figure 1) have made it possible to investigate the functions of major classes of brain cells. The retina is an ideal substrate on which to apply these techniques.1 Experimental observations indicate that the visual system of a wide range of biological creatures ranging from insects to humans can be partitioned both anatomically and functionally into

A Non-Invasive Retinal Prosthesis - Testing the Concept

We have developed a testing platform for a novel type of retinal prosthesis. Our system uses an array of light sources as non-contact stimulators. The platform consists of an imaging system based on a CMOS camera, PC based image processing, and a stimulation address system carried out on a Field Programmable Gated Array which addresses a matrix array of LEDs. Special optics are used to focus the light from the LED array onto light sensitized cells.