A Micro-Electrode Array device coupled to a laser-based system for the local stimulation of neurons by optical release of glutamate

Abstract

Optical stimulation is a promising approach to investigate the local dynamic responses of cultured neurons. In particular, flash photolysis of caged compounds offers the advantage of allowing the rapid change of concentration of either extracellular or intracellular molecules, such as neurotransmitters or second messengers, for the stimulation or modulation of neuronal activity. We describe here the use of an ultra-violet (UV) laser diode coupled to an optical fibre for the local activation of caged compounds combined with a Micro-Electrode Array (MEA) device. Local uncaging was achieved by UV irradiation through the optical fibre previously positioned by using a red laser diode. The size of the stimulation was determined using caged fluorescein, whereas its efficacy was tested by studying the effect of uncaging the neurotransmitter glutamate. Uncaged glutamate evoked neuronal responses that were recorded using either fluorescence measurements or electrophysiological recordings with MEAs, thus showing the ability of our system to induce local neuronal excitation. This method allows overcoming the limitations of the MEA system related to unfocused electrical stimulation and induction of electrical artefacts. In addition, the coupling of a UV laser diode to an optical fibre allows a precise local stimulation and a quick change of the stimulation point.

Introduction

Micro-Electrode Array devices (MEAs) have been proposed more than thirty years ago (Thomas et al., 1972, Gross, 1979, Pine, 1980) for the study of excitable cells. In these years MEA biochips have been exploited with various neuronal preparations such as dissociated cells (Martinoia et al., 2005), organotypic cultures (Egert et al., 1998) and acute tissue slices (Egert et al., 2002) for several purposes, including the investigation of neuronal plasticity and information processing in neuronal networks. Recently MEAs have been also applied as in vitro sensors to monitor both acute and chronic effects of drugs and toxins on neurons (Stett et al., 2003, Xiang et al., 2007). Thus, MEAs represent an emerging technology for the study of the functional activity of neuronal preparations.

Despite the great advantage of the MEA technology in recording extracellular activity, its applicability to cell culture/tissue stimulation presents some important limits related to the use of electrical stimulation in a conductive volume. Its major limits are the presence of large stimulus artefacts and the poorly controlled spread of electrical stimuli in the medium. Although some of the problems of stimulus artefacts have been recently solved using blanking circuits (Jimbo et al., 2003) and algorithms (Wagenaar and Potter, 2002), the spreading of electrical signals remains a limitation of MEA technology. In fact, it has been demonstrated that electrical stimuli spread to the whole biological preparation with an amplitude decreasing with the square of the distance from stimulation site (Heuschkel et al., 2002).

To overcome these limitations, an alternative approach based on optical technologies can be coupled to the MEA technology as tools for the stimulation of neurons. Among all the methods proposed to stimulate neurons with light (for reviews, see Callaway and Yuste, 2002), the use of caged compounds seems to be a powerful approach for the coupling of light with either neuronal excitation, e.g. with caged neurotransmitters, or modulation, e.g. with caged intracellular second messengers (Nerbonne, 1996).

Using caged compounds, a rapid increase in the concentration of the desired molecule can be obtained by switching the caged analogue into its active form through the cleavage of its blocking group by ultra-violet (UV) light pulses (Gurney and Lester, 1987). Since the uncaging process is spatially confined to the illuminated region, the problem related to a large spread of the stimulus can be overcome. Indeed, in the last decade various methodologies have been exploited for the efficient stimulation of neurons by using caged compounds. Traditionally, flash lamps have been coupled to epifluorescence microscopes to direct short light flashes to discrete portions of biological specimen (Kötter et al., 1998). As a technical improvement, the laser scanning photostimulation approach has been also proposed (Katz and Dalva, 1994) and widely used in uncaging experiments (Dalva and Katz, 1994, Weiler et al., 2008). Other solutions have been also proposed as uncaging tools, for example optical fibres have been coupled to arc lamps in order to spatially restrict the activation of compounds by reducing the fibre's tips (Godwin et al., 1997) and to simplify the positioning task by moving the optical fibre with a micro-manipulator (Parpura and Haydon, 1999).

Here we describe the use of a commercially available low-cost UV laser diode coupled to a small core optical fibre as a tool to achieve optical activation of caged compounds with a precise spatial and temporal control. Our stimulation tool is combined with a MEA device to detect the optically evoked electrical activity in the network, thus providing a tool to overcome the problems of the stimulus spread and artefact affecting the MEA-based stimulations. Optically evoked neuronal responses have been monitored using two independent approaches, the first based on Ca²⁺ imaging and the second based on MEA recordings.