377 The use of landmarks for navigation has been demonstrated to employ retinotopic matching, in which a series of previously memorized images are retinotopically matched with current scenes (Cartwright and Collett, 1982; Dill et al., 1993; Judd and Collett, 1998). Place learning in mammals is evidenced by the activity of hippocampal neurons that fire only when the animal is at a specific position in its learned environment (Barnes et al., 1997; Poe et al., 2000). There is evidence that cockroaches have comparable abilities because they can learn to relate distant visual cues with hidden targets (Mizunami et al., 1998) in a manner that is similar to place memory behavior of mammals (Morris, 1984). However, little is known about the underlying neural mechanisms and brain regions that support such spatial learning abilities in insects. This is in part due to the fact that few behavioral paradigms are available for studying spatial learning in an immobilized animal that might then be subjected to intracellular recording, as is done on rodents. The antennal motor system of insects can be used to develop novel behavioral paradigms for studying associative memory (Lent and Kwon, 2004) and, by extension, spatial memory. Antennal motor actions can be elicited by different modalities, including olfactory, tactile and visual stimuli (Erber et al., 1993; Erber and Pribbenow, 1997). Antennal movements elicited by visual stimuli demonstrate active sensory exploration, such as by restrained honey bees that move their antennae towards the direction of a moving grating (Erber and Pribbenow, 1997). Visual inputs have also been shown to elicit antennal movements in crickets (Honegger, 1981). Such behaviors have been utlized by experiments in which animals were operantly conditioned to extend their antennae towards a target in order to receive a reward (Erber et al., 1993; Kisch and Erber, 1999). In nature, directed antennal movements that are elicited by a sensory stimulus (here termed antennal projection responses or APRs) may be employed to locate an olfactory stimulant, such as the odor of food, a conspecific or a predator (Bell, 1981). As shown previously, APRs can be conditioned to point to a visual cue after its learned association with a food odor (Lent and Kwon, 2004). The present study describes a novel visual association paradigm to demonstrate spatial learning on restrained cockroaches, again exploiting antennal movements as an indicator of learning. The present results show that restrained insects can learn to recognize spatial relationships between distant cues. Because this can be demonstrated on an immobilized animal, the results provide a crucial step towards investigating place memory at the level of defined circuitry. Materials and methods Animals Male American cockroaches (Periplaneta americana L.) raised in a laboratory colony maintained on water and cat food (IAMS, Dayton, Ohio) were used in the behavioral experiments. Cockroaches were kept at 25±1°C on a 12·h:12·h light:dark cycle. Test animals were isolated from colonies. The Journal of Experimental Biology 207, 377-383 Published by The Company of Biologists 2004 doi:10.1242/jeb.00737 Spatial learning abilities were tested in restrained cockroaches by observing antennal projection responses towards the positions of a learned visual cue perceived monocularly by one eye in the context of a second stimulus provided to the contralateral eye. Memory of the position of the conditioning stimulus relative to the contralateral reference stimulus was tested by altering the relative positions of the two stimuli. Memory of the conditioning stimulus is retained if the angle between the conditioning stimulus and the contralateral reference stimulus is maintained. The results suggest that during learning the insect recognizes spatial relationships between the conditioning stimulus and the contralateral reference stimulus. Possible mechanisms, such as retinotopic matching versus angular matching, are discussed. Key words: place memory, cockroach, Periplaneta americana, antennal movement, behavior. Summary Introduction Spatial learning in the restrained American cockroach Periplaneta americana Hyung-Wook Kwon 1, *, David D. Lent 2, * ,† and Nicholas J. Strausfeld 2 1 Dept of Biological Sciences, 6270 Medical Research Building III, Vanderbilt University, 465 21st Ave. South, Nashville, TN 37235, USA and 2 Arizona Research Laboratories, Division of Neurobiology, 611 Gould-Simpson Building, PO Box 210077, The University of Arizona, Tucson, AZ 85721, USA *These two authors contributed equally to this work † Author for correspondence (e-mail: dlent@u.arizona.edu) Accepted 29 September 2003