No abstract

The circadian clock enables organisms to anticipate daily environmental cycles and drives corresponding changes in behavior [1, 2]. Such endogenous oscillators also enable animals to display time-specific memory [1, 3-5]. For instance, mice and honeybees associate the location of a stimulus (like food or mate) with a certain time of day (time-place learning) [6, 7]. However, the mechanism underlying time-related learning and memory is not known. In the present study, we investigate time-specific odor learning. We use a genetically tractable animal, the fly Drosophila melanogaster. Starved flies are trained in the morning and afternoon to associate distinct odors with sucrose reward. The training is repeated the next day, and their time-dependent odor preference is tested on the third day. Our results indicate that Drosophila can express appetitive memory at the relevant time of day if the two conditioning events are separated by more than 4 hr. Flies can form time-odor associations in constant darkness (DD) as well as in a daily light-dark (LD) cycle, but not when kept under constant light (LL) conditions. Circadian clock mutants, period(01) (per(01)) and clock(AR) (clk(AR)), learned to associate sucrose reward with a certain odor but were unable to form time-odor associations. Our findings show that flies can utilize temporal information as an additional cue in appetitive learning. Time-odor learning in flies depends on a per- and clk-dependent endogenous mechanism that is independent of environmental light cues.

Starvation causes a motivational state that facilitates diverse behaviors such as feeding, walking, and search. Starved can form odor/feeding-time associations but the role of starvation in encoding of "time" is poorly understood. Here we show that the extent of starvation is correlated with the fly's ability to establish odor/feeding-time memories. Prolonged starvation promotes odor/feeding-time associations after just a single cycle of reciprocal training. We also show that starvation is required for acquisition but is dispensable for retrieval of odor/feeding-time memory. Finally, even with extended starvation, a functional circadian oscillator is indispensable for establishing odor/feeding-time memories.

Sleep is a universally conserved physiological state which contributes toward basic organismal functions, including cognitive operations such as learning and memory. Intriguingly, organisms can sometimes form memory even without sleep, such that Drosophila display sleep-dependent and sleep-independent memory in an olfactory appetitive training paradigm. Sleep-dependent memory can be elicited by the perception of sweet taste, and we now show that a mixed-sex population of flies maintained on sorbitol, a tasteless but nutritive substance, do not require sleep for memory consolidation. Consistent with this, silencing sugar-sensing gustatory receptor neurons in fed flies triggers a switch to sleep-independent memory consolidation, whereas activating sugar-sensing gustatory receptor neurons results in the formation of sleep-dependent memory in starved flies. Sleep-dependent and sleep-independent memory relies on distinct subsets of reward signaling protocerebral anterior medial dopaminergic neurons (PAM DANs) such that PAM-b92mp DANs mediate memory in fed flies whereas PAM-a1 DANs are required in starved flies. Correspondingly, we observed a feeding-dependent calcium increase in PAM-b92mp DANs, but not in PAM-a1 DANs. Following training, the presence of sweet sugars recruits PAM-b92mp DANs, whereas tasteless medium increases calcium in PAM-a1 DANs. Together, this work identifies mechanistic underpinnings of sleep-dependent memory consolidation, in particular demonstrating a role for the processing of sweet taste reward signals.