A method for recording behavior and multineuronal CNS activity from tethered insects flying in virtual space John R. Gray *, Vincent Pawlowski, Mark A. Willis 1 Arizona Research Laboratories Division of Neurobiology, 611 Gould-Simpson Building, University of Arizona, Tucson, AZ 85721, USA Received 2 January 2002; received in revised form 16 July 2002; accepted 17 July 2002 Abstract We describe a low cost, novel virtual reality-based insect flight simulator that combines visual, olfactory and mechanosensory stimuli with multichannel neurophysiological recording techniques. Three-dimensional visual environments were created using customized modifications of a first person flight simulator computer game. Experiments could be performed in open-loop, where the flying insect’s movement through the environment is ‘driven’ by the human operator, or in closed-loop where the movement of the environment is controlled by optically sensed movements of the insect’s abdomen. During flight, we recorded multineuronal activity from the ventral nerve cord between the brain and thoracic ganglia. Results show that in open-loop conditions, induced turns of the environment evoked characteristic compensatory optomotor responses. Coordination of wing and body kinematics was similar to that observed in free flight. In closed-loop conditions, the insect was able to navigate through the simulated environment and produce flight tracks in response to presentation of pheromone that resemble those observed in free flight. We discuss the effectiveness of this preparation and its utility for addressing specific questions of insect flight as well as general questions in neuroethology. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Flight simulator; Insect; Multineuronal recording; Multimodal stimuli; Neuroethology; Virtual reality 1. Introduction The main goal of neuroethology is to understand how the nervous system produces and coordinates adaptive behaviors within the animal’s natural environment. To this end many investigations have studied specific components of the nervous system to identify mechan- isms underlying: (1) stimulus detection and perception, (2) sensory motor integration and (3) motor output that produces a certain behavior. Due to limitations of available techniques these components were studied individually. Many important discoveries have come from these types of studies; however, the goal of incorporating these three components into an experi- ment in which an animal is free to locomote within a natural environment has been achieved in only a few select systems. Studies of insect flight have been instrumental in extending our understanding of how stimuli of different modalities are related to production of adaptive beha- vior. Many investigators have developed flight simula- tors designed to present the insect with controlled visual stimuli while recording behavioral and/or neurophysio- logical activity. These include studies on locusts (Baker, 1979; Reichert et al., 1985; Thu ¨ ring, 1986; Robert, 1988; Mo ¨hl, 1988; Hensler and Robert, 1990; Miall, 1990; Rowell and Reichert, 1991; Baader, 1991; Robertson and Johnson, 1993), flies (Heisenberg and Wolf, 1979; Zanker et al., 1991; Kimmerle et al., 2000; Kern et al., 2001) and moths (Olberg and Willis, 1990; Kanzaki et al., 1991; Vickers and Baker, 1994). Indeed, there are descriptions of flight simulators that produce interactive visual stimulation which is controlled either by move- ment of the insect or by physiological activity of muscles or neurons (Robert, 1988; Mo ¨hl, 1988; Kimmerle et al., 2000). These studies examined behavioral responses or responses of individual neurons to visual stimuli in * Corresponding author. Address: Department of Biology, 112 Science Place, University of Saskatchewan, Saskatoon, Sask., Canada S7N 5E2. Tel.:  /1-306-966-7305; fax:  /1-306-966-4461 E-mail address: jack.gray@usask.ca (J.R. Gray). 1 Department of Biology, Case Western Reserve University, 10900 Euclid Boulevard, Cleveland, OH 44106, USA Journal of Neuroscience Methods 120 (2002) 211 /223 www.elsevier.com/locate/jneumeth 0165-0270/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII:S0165-0270(02)00223-6