Brief Communication Olfactory conditioning of the sting extension reflex in honeybees: Memory dependence on trial number, interstimulus interval, intertrial interval, and protein synthesis Martin Giurfa, 1,2,4 Eve Fabre, 1,2,3 Justin Flaven-Pouchon, 1,2,3 Helga Groll, 1,2,3 Barbara Oberwallner, 1,2,3 Vanina Vergoz, 1,2,3 Edith Roussel, 1,2 and Jean Christophe Sandoz 1,2 1 Universite ´ de Toulouse, UPS, Centre de Recherches sur la Cognition Animale, F-31062 Toulouse Cedex 9, France; 2 CNRS, Centre de Recherches sur la Cognition Animale, F-31062 Toulouse Cedex 9, France Harnessed bees learn to associate an odorant with an electric shock so that afterward the odorant alone elicits the sting extension response (SER). We studied the dependency of retention on interstimulus interval (ISI), intertrial interval (ITI), and number of conditioning trials in the framework of olfactory SER conditioning. Forward ISIs (conditioned stimulus [CS] before unconditioned stimulus [US]) supported higher retention than a backward one (US before CS) with an optimum around 3 sec. Spaced trials (ITI 10 min) supported higher retention than massed trials (ITI 1 min) and led to the formation of a late long-term memory (l-LTM) that depended on protein synthesis. Our results reaffirm olfactory SER conditioning as a reliable tool for the study of learning and memory. The honeybee Apis mellifera is a main invertebrate model for the study of learning and memory as it allows combining controlled conditioning protocols with a simultaneous access to the nervous system in the laboratory (Menzel 1999; Giurfa 2007). The main protocol used to this end relies on the proboscis extension reflex (PER), the appetitive reflex exhibited by a harnessed honeybee to a sugar reward (the unconditioned stimulus, or US) delivered to its antennae and mouthparts. After appropriate pairing of an odorant (the conditioned stimulus, or CS) with sucrose presentations, the bee learns to associate odorant and sugar reward so that the odorant alone elicits PER (Takeda 1961; Bitterman et al. 1983). Despite the important progress made in understanding the be- havioral, cellular, and molecular bases of this appetitive learning (Menzel 1999; Giurfa 2007), until recently it has been impossible to study aversive learning in bees in such a way that behavioral records would be accompanied by access to the nervous system. This gap has been filled by a novel conditioning protocol in which individually harnessed bees learn to associate an initially neutral odorant (CS) with a mild electric shock (US) (Vergoz et al. 2007). Bees fixed individually on a metallic holder (Fig. 1A) reflexively extend their sting (sting extension response, or SER) upon delivery of an electric shock to the thorax (Nu ´n ˜ez et al. 1997; Balderrama et al. 2002), thus showing a typical defensive response to poten- tially noxious stimuli (Breed et al. 2004). After successful condi- tioning, the odorant elicits SER, a conditioned response that can be retrieved 1 h after conditioning (Vergoz et al. 2007). This form of conditioning is indeed aversive as shown by the fact that bees trained in this way and transferred to the operant context of a Y-maze, where they can freely walk and choose between the shock-associated odor and a non-shock-associated odor, explicitly avoid the punished odor and choose the non-shock-associated odor 1 h after conditioning (Carcaud et al. 2009). Classical features of Pavlovian conditioning protocols such as the dependence of retrieval on the interstimulus interval (ISI; the interval between CS and US onset) and intertrial interval (ITI; the interval between consecutive trial onset) have not been analyzed thus far in olfactory SER conditioning. Furthermore, although olfactory SER conditioning leads to an aversive memory that can be retrieved either in a Pavlovian (Vergoz et al. 2007) or in an operant framework (Carcaud et al. 2009) 1 h after conditioning, it is unknown whether it leads to the formation of robust LTMs, retrievable some days after conditioning. In the honeybee, one pairing of an odorant with sucrose (i.e., one conditioning trial) leads to an early long-term memory (e-LTM) that can be retrieved 24 to 48 h after conditioning. Three conditioning trials, on the other hand, lead to a stable late long-term memory (l-LTM) that can be retrieved 72 h or more after conditioning. Although the presence of short-term memories (STM) and medium-term mem- ories (MTM)—retrievable just after conditioning or 1 h after it, respectively—have been shown in olfactory SER conditioning (Vergoz et al. 2007; Carcaud et al. 2009), no evidence thus far supports the presence of LTM after this form of learning. Here we performed a series of experiments aimed at characterizing the effect of variables such as ISI and ITI on learning and retention performance in olfactory SER conditioning. We asked whether this protocol leads to the formation of LTM and whether such a memory is protein-synthesis dependent. Honeybee foragers were captured when leaving the hive. These individuals have a fully developed sting reflex (Burrell and Smith 1994) and exhibit higher shock responsiveness and aver- sive learning scores (Roussel et al. 2009). In all experiments, we followed the standard criterion used in olfactory appetitive PER conditioning, which consists of checking before conditioning and after retention tests that the unconditioned response (SER) to the US (the electric shock) is intact. This control ensures that bees have 3 These authors contributed equally to this work. 4 Corresponding author. Email giurfa@cict.fr; fax 33-561-55-61-54. Article is online at http://www.learnmem.org/cgi/doi/10.1101/lm.1603009. 16:761–765 Ó 2009 Cold Spring Harbor Laboratory Press 761 Learning & Memory ISSN 1072-0502/09; www.learnmem.org Cold Spring Harbor Laboratory Press on December 22, 2009 - Published by learnmem.cshlp.org Downloaded from