Invertebrate learning and memory: Fifty years of olfactory conditioning of the proboscis extension response in honeybees

Abstract

The honeybee Apis mellifera has emerged as a robust and influential model for the study of classical conditioning, thanks to the existence of a powerful Pavlovian conditioning protocol, the olfactory conditioning of the proboscis extension response (PER). In 2011, the olfactory PER conditioning protocol celebrates 50 years since it was first introduced by Kimihisa Takeda in 1961. Here, we review its origins, developments, and perspectives in order to define future research avenues and necessary methodological and conceptual evolutions. We show that olfactory PER conditioning has become a versatile tool for the study of questions in extremely diverse fields in addition to the study of learning and memory and that it has allowed behavioral characterizations, not only of honeybees, but also of other insect species, for which the protocol was adapted. We celebrate, therefore, Takeda's original work and prompt colleagues to conceive and establish further robust behavioral tools for an accurate characterization of insect learning and memory at multiple levels of analysis.Classical conditioning (Pavlov 1927) is a form of conditioning in which a subject learns to associate a neutral stimulus (the "conditioned stimulus," or CS), which does not originally elicit a behavioral response, with a stimulus of biological significance (the "unconditioned stimulus," or US), which elicits an innate, often reflexive, response. Through this association, the originally neutral stimulus acquires the capacity to elicit a conditioned response.Decades of research on animal learning and memory have established some invertebrates (e.g., the sea hare, Aplysia californica, the fruit fly, Drosophila melanogaster, the honeybee, Apis mellifera) as standard models for the study of classical conditioning (Giurfa 2007b;Menzel et al. 2007). This success can be attributed to the fact that these animals can learn nonassociative as well as Pavlovian and operant associations and possess relatively simple nervous systems that allow retracing of these phenomena to the cellular and molecular levels in different kinds of laboratory preparations. Among these invertebrates, the honeybee, Apis mellifera, has emerged as a robust and influential model for the study of

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“…For over 50 years an assay for evaluating olfactory conditioning of the proboscis extension reflex (PER) has been used as a reliable method for evaluating appetitive learning and memory in honey bees (Guirfa and Sandoz, 2012). These experiments pair a conditioned stimulus (e.g., an odor) with an unconditioned stimulus (e.g., sucrose) provided immediately afterward, which elicits the proboscis extension (Menzel, 2012).…”

“…After conditioning, the odor alone will lead to the conditioned PER. This methodology has aided in the elucidation of five types of olfactory memory phases in honey bee, which include early short-term memory, late short-term memory, mid-term memory, early long-term memory, and late long-term memory (Guirfa and Sandoz, 2012). These phases are dependent on the type of conditioned stimulus, the intensity of the unconditioned stimulus, the number of conditioning trials, and the time between trials.…”

“…These phases are dependent on the type of conditioned stimulus, the intensity of the unconditioned stimulus, the number of conditioning trials, and the time between trials. Where formation of short-term memory occurs minutes after conditioning and decays within minutes, memory consolidation or stabilization of a memory trace after initial acquisition leads to mid-term memory, which lasts 1 d and is characterized by activity of the cAMP-dependent PKA (Guirfa and Sandoz, 2012). Multiple conditioning trials increase the duration of the memory after learning and coincide with increased Ca 2+ -calmodulin-dependent PKC activity (Guirfa and Sandoz, 2012).…”

“…Where formation of short-term memory occurs minutes after conditioning and decays within minutes, memory consolidation or stabilization of a memory trace after initial acquisition leads to mid-term memory, which lasts 1 d and is characterized by activity of the cAMP-dependent PKA (Guirfa and Sandoz, 2012). Multiple conditioning trials increase the duration of the memory after learning and coincide with increased Ca 2+ -calmodulin-dependent PKC activity (Guirfa and Sandoz, 2012). Early long-term memory, where a conditioned response can be evoked days to weeks after conditioning requires translation of existing mRNA, whereas late long-term memory requires de novo gene transcription and can last for weeks (Guirfa and Sandoz, 2012).…”

“…Multiple conditioning trials increase the duration of the memory after learning and coincide with increased Ca 2+ -calmodulin-dependent PKC activity (Guirfa and Sandoz, 2012). Early long-term memory, where a conditioned response can be evoked days to weeks after conditioning requires translation of existing mRNA, whereas late long-term memory requires de novo gene transcription and can last for weeks (Guirfa and Sandoz, 2012). Knowledge gleaned from PER assays has not only increased neurobiological understanding of memory but has also provided a method for assessing the effects of chemical perturbation on learning and memory in honey bees.…”