369 Although Drosophila has provided valuable insights into the genetics of learning and memory (Tully and Quinn, 1985; Dubnau et al., 2001), it has so far been practically intractable for physiological studies of identified neurons and neuronal circuits. Even the honey bee (Apis mellifera), which has yielded much information about behaviors associated with olfactory learning and memory, has yielded only three studies suggesting learning-associated changes in neuronal physiology (Hammer, 1993; Mauelshagen, 1993; Grunewald, 1999). In comparison, the cockroach Periplaneta americana is demonstrably resilient to long-term intra- and extracellular studies of identified neurons and circuits (Mizunami et al., 1998; Li and Strausfeld, 1997; Strausfeld and Li, 1999) and has been shown to possess mammalian-like place memory mediated by its mushroom bodies (Mizunami et al., 1998). While behavioral studies on cockroaches have demonstrated their suitability for learning and memory studies (Balderrama, 1980; Gadd and Raubenheimer, 2000), a valid argument against using this taxon is that the behavioral paradigms have been designed for free-moving animals and are thus unacceptable for studies at the cellular level. In this and the succeeding paper (Kwon et al., 2004), we describe learning paradigms that have been developed for use on restrained animals so that, as in the case of the honey bee’s proboscis extension reflex, these can be employed for intracellular and biochemical studies. Experiments described here rely on a stereotyped foraging behavior. This is the antennal projection response (APR), which is reminiscent of sniffing in mammals (Gray and Skinner, 1988) or antennular flicking in crayfish and spiny lobsters (Mellon, 1997; Derby, 2000). Such actions are used to assess a continuously changing olfactory milieu and provide the brain with data for locating smells. In lobsters, the frequency and directional control of antennular flicking behaviors increase as mixtures of odor components increase (Mellon, 1997). Other modalities can also trigger antennal projection responses. For example, in honey bees, antennal scanning can be elicited by visual, olfactory and mechanical cues (Erber et al., 1993), and antennal movements can be operantly conditioned (Kisch and Erber, 1999). When crickets track moving objects, their antennae move in the same direction as the object (Honegger, 1981). Here, we describe experiments that demonstrate a plastic behavior that can be driven in immobilized cockroaches. The behavior, which is expressed by APRs towards an olfactory stimulus source, can be classically conditioned and can be used for studying spatial context in learning and memory. We describe classical conditioning of APRs towards a neutral stimulus [a green light cue (conditioned stimulus, CS)] coupled with an odor source (unconditioned stimulus, US). The classical conditioning results in an APR towards the green light cue (CS), mimicking the response towards an odor source The Journal of Experimental Biology 207, 369-375 Published by The Company of Biologists 2004 doi:10.1242/jeb.00736 Using antennal movements as an indicator of learning and retention, an associative learning paradigm has been developed to investigate associative memory between visual and olfactory stimuli. Experiments were performed on the restrained cockroach Periplaneta americana, which normally moves its antennae towards a localized odor source. Such ‘antennal projection responses’ (APRs) are exploited to demonstrate long-term memory, where an APR is elicited by a conditioned stimulus (CS; green light point source) paired with a spatially coincident odor [the unconditioned stimulus (US)]. Association of the CS with the US is established after five trials. Before training, a visual cue alone does not elicit an APR. This behavior is elicited by a visual cue only after pairing it with an odor stimulus. The acquired APR to the green light cue persists for up to 72·h, indicative of long-term memory. This paradigm is thus suitable for future studies of neural correlates of learning and memory on restrained animals. Key words: behavior, insect, cockroach, Periplaneta americana, memory, multimodal integration, antennal movement. Summary Introduction Antennal movements reveal associative learning in the American cockroach Periplaneta americana David D. Lent 1, * and Hyung-Wook Kwon 2 1 Arizona Research Laboratories, Division of Neurobiology, 611 Gould-Simpson Building, PO Box 210077, The University of Arizona, Tucson, AZ 85721, USA and 2 Dept of Biological Sciences, 6270 Medical Research Building III, Vanderbilt University, 465 21 st Ave. South, Nashville, TN 37235, USA *Author for correspondence (e-mail: dlent@u.arizona.edu) Accepted 29 September 2003