Journal of Neuroscience Methods 175 (2008) 44–57 Contents lists available at ScienceDirect Journal of Neuroscience Methods journal homepage: www.elsevier.com/locate/jneumeth A novel control software that improves the experimental workflow of scanning photostimulation experiments Michael H.K. Bendels a,b,c,∗,1 , Prateep Beed a,1 , Christian Leibold b,c , Dietmar Schmitz a,d,1 , Friedrich W. Johenning a,1 a NeuroScience Research Center, Charité, Universitätsmedizin Berlin, Charité-Platz 1, 10117 Berlin, Germany b Department of Biology II, University of Munich, Division of Neurobiology, Großhaderner Str. 2, 82152 Planegg-Martinsried, Germany c Bernstein Center for Computational Neuroscience Munich, Department of Neurology, University Hospital Munich-Grosshadern, Ludwig-Maximilians University Munich, Marchioninistr. 15, 81377 Munich, Germany d Bernstein Center for Computational Neuroscience Berlin, Institute of Biology, Humboldt-University Berlin, Philippstr. 13, Haus 6, 10115 Berlin, Germany article info Article history: Received 27 February 2008 Received in revised form 25 July 2008 Accepted 1 August 2008 Keywords: Photostimulation Uncaging Functional anatomy Stellate cell Pyramidal cell Entorhinal cortex abstract Optical uncaging of caged compounds is a well-established method to study the functional anatomy of a brain region on the circuit level. We present an alternative approach to existing experimental setups. Using a low-magnification objective we acquire images for planning the spatial patterns of stimulation. Then high-magnification objectives are used during laser stimulation providing a laser spot between 2 m and 20 m size. The core of this system is a video-based control software that monitors and controls the con- nected devices, allows for planning of the experiment, coordinates the stimulation process and manages automatic data storage. This combines a high-resolution analysis of neuronal circuits with flexible and effi- cient online planning and execution of a grid of spatial stimulation patterns on a larger scale. The software offers special optical features that enable the system to achieve a maximum degree of spatial reliability. The hardware is mainly built upon standard laboratory devices and thus ideally suited to cost-effectively complement existing electrophysiological setups with a minimal amount of additional equipment. Finally, we demonstrate the performance of the system by mapping the excitatory and inhibitory connections of entorhinal cortex layer II stellate neurons and present an approach for the analysis of photo-induced synaptic responses in high spontaneous activity. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Scanning UV-photostimulation, originally introduced by Katz and Dalva (1994), has become a well-established method in neu- rophysiology (Callaway and Yuste, 2002). Its application ranges from the analysis of neuronal connectivity of local brain circuits (Brivanlou et al., 2004; Jin et al., 2006; Lam et al., 2006; Schubert et al., 2001, 2003, 2006; Shepherd et al., 2003) and the func- tional characterization of synapses (Godwin et al., 1997; Kandler et al., 1998) to measurements of the distribution of specific recep- tors on the surface of neurons (Dodt et al., 1998; Frick et al., 2001) and the investigation of dendritic integration (Shoham et al., 2005). Despite this wide scope of applications, the common idea shared by all photostimulation experiments is the coordinated ∗ Corresponding author at: Bernstein Center for Computational Neuroscience Munich, Department of Biology II, University of Munich, Großhaderner Str. 2, 82152 Planegg-Martinsried, Germany. Tel.: +49 89 2180 74355; fax: +49 89 2180 74304. E-mail address: bendels@zi.biologie.uni-muenchen.de (M.H.K. Bendels). 1 These authors contributed equally to this work. spatio-temporal conversion of an inactive ‘caged’ transmitter to an activated ‘uncaged’ transmitter by a transient and highly energetic mostly ultraviolet light beam, while simultaneously recording from one or more neurons. Since photolytic activation provides a fast way to produce perturbations in a chemical system without using an invasive mechanical instrumentation (Lester and Nerbonne, 1982), it is perfectly suited to analyze neuronal networks and their com- plex interactions. For investigations of the qualitative assessment of connectivity patterns between brain regions (Dantzker and Callaway, 2000), a high resolution of photostimulation in the range below 10 m is not critical. In contrast, for mapping experiments aiming to quanti- tatively estimate synaptic conductances, the diameter of the laser spot has to be decreased to permit for finer stimulation patterns (Dodt et al., 1998). Stimulation of neurons at the single-spine level, where the focal uncaging volume needs to be small enough to mimic glutamate release in the synaptic cleft, can only be achieved using a two-photon-laser based uncaging system (Losonczy and Magee, 2006; Matsuzaki et al., 2004). However, larger focal uncaging volumes inherent to one-photon based uncaging systems are sufficient to investigate subcellular 0165-0270/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2008.08.010