Classical Conditioning of the Proboscis Extension Reflex in the Honeybee

Abstract

Insects have sophisticated learning abilities subtended by simple neural systems and lower numbers of neurons compared to vertebrates. Especially, honeybees (Apis mellifera) are reported to have the highest and broad range of learning abilities. In this chapter, we introduce a classic behavioral tool for the study of olfactory learning and memory in bees, the olfactory classical conditioning of the proboscis extension reflex (PER). In this protocol, individually harnessed honeybees are trained to associate an odor with sucrose solution. The resulting olfactory learning is fast and induces robust olfactory memories that have been characterized at the behavioral, neuronal, and molecular levels. We detail step-by-step the methodology of olfactory PER conditioning in order to provide a standardized framework for experiments using this tool. We also review research highlights revealed by olfactory conditioning of PER and variations of this procedure applied in the case of honeybees.

Appendix A Column: History of a Classical Conditioning of PER in Honeybees

Long before research on PER conditioning started, it was well known that the PER could be elicited by stimulating gustatory organs like the antennae, tarsi, or mouthparts with sugar solution. The PER had thus been detected in bees (Frings 1944; Frings and Frings 1949), flies (Minnich 1926), and butterflies (Minnich 1921), among others. Later, a Japanese researcher who had worked with Karl von Frisch, Masutaro Kuwabara, realized that this appetitive reflex could be conditioned using visual stimuli as CS and sucrose solution delivered to the tarsi as US (Kuwabara 1957). However, Kuwabara’s work did not receive broad attention as shown by the fact that almost 50 years had to pass before other researchers published results on honeybee visual conditioning using Kuwabara’s method (Hori et al. 2006; Hori et al. 2007). For this conditioning to work, Kuwabara and Hori et al. had to cut the bees’ antennae. The low acquisition rates observed in antennae-deprived bees despite long conditioning procedures (Hori et al. 2006; Hori et al. 2007) may be related to this fact. It has been recently shown that antennae deprivation reduces sucrose responsiveness when measured through tarsal stimulation (de Brito Sanchez et al. 2008), probably leading to a reduction of US value and of acquisition and retention performances.

The olfactory conditioning of PER was afterwards established by a student of Kuwabara, Kimihisa Takeda, who reported on this procedure in 1961 (Takeda 1961) using odors as CS and sucrose solution as US. As was common use 50 years ago, Takeda did not report any acquisition, retention, or extinction curves, nor did he provide any statistical analysis of PER responses. He only presented tables with the raw data of single bees, with very low sample sizes. Despite data paucity, lack of statistics, absence of controls, and representative sample sizes, Takeda’s work laid down the experimental principles and the scientific questions that would serve as a basis for future, more controlled research on honeybee learning and memory. He showed for the first time extinction learning (including spontaneous recovery), stimulus generalization and discrimination, conditioned inhibition, and second-order conditioning in the olfactory domain in honeybees. In this way he established olfactory PER conditioning as a useful tool for the study of invertebrate learning and memory.