Multiple molecular links between the circadian clock and memory centers in honey bees

Time and memory are intimately linked: the capability to learn and recall varies over the day and humans and many animals can associate important events with the time of day. However, how the circadian clock and memory centers are connected is not well understood. We time-trained honey bee foragers, collected them at several different time-points for a complete circadian day and performed time-series RNA-sequencing (RNA-seq) to analyze gene expression changes in focal populations of mushroom body (MB) neurons. Thus, we identified synchronized peak-level expression of memory-related genes during training time, receptor for pigment dispersing factor, the major neuropeptide exclusively produced by some central pacemaker neurons, and expression of major clock genes that might represent local clocks. Overall design: To identify candidate molecular interactions between the clock and memory centers we used laser capture microdissection (LCM) to collect small Kenyon cell (sKC)-enriched tissue samples, the major multimodal neuronal population of the MBs and an identified locus involved in time-memory processes. We performed time-series RNA-seq with sKC-enriched samples from time-trained honey bee foragers collected at 7 different time-points over a complete circadian day. For each of the 7 different time-points, we collected two replicates each from two different colonies. The two colonies were independent and the behavioral experiments were performed separately.