Reversible Downregulation of Protein Kinase A during Olfactory Learning Using Antisense Technique Impairs Long-Term Memory Formation in the Honeybee,Apis mellifera

Abstract

In this study, we examined the role of cAMP-dependent protein kinase (PKA) in associative olfactory learning of the honeybee, Apis mellifera. In the bee, specific interference with molecules to clarify their role in a certain behavior is difficult, because genetic approaches, such as mutants or transgenic animals, are not feasible at the moment. As a new approach in insects in vivo, we report the use of short antisense oligonucleotides. We show that phosphorothioate-modified oligodeoxynucleotides complementary to the mRNA of a catalytic subunit of PKA directly injected into the bee brain cause a reversible and specific downregulation of both the amount of the catalytic subunit and of PKA activity by 10-15%. The amounts of the regulatory subunit of PKA, as well as PKC, are not affected. The slight "knockdown" of PKA activity during the training procedure, a classical olfactory conditioning of the proboscis extension reflex, neither affects acquisition nor memory retention 3 or 6 hr after training. However, it causes an impairment of long-term memory retention 24 hr after training. Downregulation of PKA 3 hr after training has no detectable effect on memory formation. We conclude that PKA contributes to the induction of a long-term memory 24 hr after training when activated during learning. Second, we demonstrate that the antisense technique is feasible in honeybees in vivo and provides a new and powerful tool for the study of the molecular basis of learning and memory formation in insects.