Journal of Neuroscience Methods 175 (2008) 70–78 Contents lists available at ScienceDirect Journal of Neuroscience Methods journal homepage: www.elsevier.com/locate/jneumeth A Micro-Electrode Array device coupled to a laser-based system for the local stimulation of neurons by optical release of glutamate Diego Ghezzi a,b,∗ , Andrea Menegon c,d , Alessandra Pedrocchi b , Flavia Valtorta c,d , Giancarlo Ferrigno b a Department of Neuroscience and Brain Technologies, The Italian Institute of Technology, via Morego 30, 16163 Genova, Italy b NeuroEngineering and Medical Robotics Laboratory, Bioengineering Department, Politecnico di Milano, p.zza Leonardo da Vinci 32, 20133, Milano, Italy c San Raffaele Scientific Institute and Vita-Salute University, via Olgettina 58, 20132 Milano, Italy d Unit of Molecular Neuroscience, The Italian Institute of Technology, via Olgettina 58, 20132 Milano, Italy article info Article history: Received 7 March 2008 Received in revised form 1 August 2008 Accepted 4 August 2008 Keywords: Caged compound Uncaging Optical fiber Flash photolysis MEA Neuronal excitation abstract Optical stimulation is a promising approach to investigate the local dynamic responses of cultured neu- rons. 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 neuro- transmitter 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. © 2008 Elsevier B.V. All rights reserved. 1. 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 dissoci- ated 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 stimula- ∗ Corresponding author at: Department of Neuroscience and Brain Technologies, The Italian Institute of Technology, via Morego 30, 16163 Genova, Italy. Tel.: +39 010 71781 524. E-mail address: diego.ghezzi@iit.it (D. Ghezzi). tion presents some important limits related to the use of electrical stimulation in a conductive volume. Its major limits are the pres- ence 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 technol- ogy. 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 pro- posed 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 0165-0270/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2008.08.003