Circadian regulation of insect olfactory learning

Abstract

Olfactory learning in insects has been used extensively for studies on the neurobiology, genetics, and molecular biology of learning and memory. We show here that the ability of the cockroach Leucophaea maderae to acquire olfactory memories is regulated by the circadian system. We investigated the effect of training and testing at different circadian phases on performance in an odor-discrimination test administered 30 min after training (short-term memory) or 48 h after training (long-term memory). When odor preference was tested by allowing animals to choose between two odors (peppermint and vanilla), untrained cockroaches showed a clear preference for vanilla at all circadian phases, indicating that there was no circadian modulation of initial odor preference or ability to discriminate between odors. After differential conditioning, in which peppermint odor was associated with a positive unconditioned stimulus of sucrose solution and vanilla odor was associated with a negative unconditioned stimulus of saline solution, cockroaches conditioned in the early subjective night showed a strong preference for peppermint and retained the memory for at least 2 days. Animals trained and tested at other circadian phases showed significant deficits in performance for both short- and long-term memory. Performance depended on the circadian time (CT) of training, not the CT of testing, and results indicate that memory acquisition rather than retention or recall is modulated by the circadian system. The data suggest that the circadian system can have profound effects on olfactory learning in insects.

Fig. 1.

Long-term learning and memory depends on the circadian phase. The graphs plot the distribution of PPI expressed by individuals either before (A and C) or after (B and D) conditioning. Animals were isolated in constant darkness 3 days before the first preference test. (A) Distribution of 23 naive animals tested for preference for peppermint or vanilla at CT 14 on day 3 of constant darkness. The clustering near the origin shows the strong preference for vanilla. These animals underwent conditioning at CT 14 on day 5 of constant darkness and were tested again on day 7. (B) Results of the preference test 48 h after conditioning. There is a strong preference for peppermint, indicating learning and long-term memory. (C and D) Same sequence of testing, training, and retesting at CT 2 for a group of 19 animals. There is no significant change in the preference for vanilla, indicating a profound deficit at this phase of the circadian cycle.

Fig. 2.

Circadian regulation of long-term olfactory learning and memory in the cockroach. (A) Mean PPI (±SE) for naive animals tested at CT 2 (n = 19), CT 8 (n = 23), CT 14 (n = 23), and CT 20 (n = 21). All groups showed a strong preference for vanilla, indicating that the ability to distinguish between the odors is independent of the circadian phase. (B) Mean PPI when animals were retested 48 h after training. (C) Change in PPI between the pre- and posttraining tests. Only those animals trained and tested at CT 14 showed a significant increase in the preference for peppermint (Kruskal–Wallis one-way ANOVA, P < 0.0001). Significant differences between groups (P < 0.05, Dunn's post hoc analysis) are indicated by differences in letters at the top of each bar. The results indicate that there is a circadian modulation of long-term olfactory learning and memory. (D) Percentage of saline or sucrose consumed at either CT 2 or CT 14. There is no circadian variation in the response to the US.

Fig. 3.

The circadian phase of training is more important than the phase of testing. (A) Change in PPI for animals tested at CT 2, trained at CT 14, and retested at CT 2, 36 h after training (n = 19), compared with animals tested at CT 14, trained at CT 2, and retested at CT 14, 36 h after training (n = 24). (B) PPI for animals tested before training at CT 14 and retested 36 h after training (CT 2) and again 48 h after training (CT 14). In both of the posttraining tests, the animals showed a significant increase in preference for peppermint (P < 0.0001), and there no significant difference between the two posttraining test times. The data indicate that recall/performance is independent of CT of testing and only depends on the CT of training.

Fig. 4.

Circadian regulation of short-term olfactory learning in the cockroach. (A) Mean PPI (± SE) for naive animals tested at CT 2 (n = 26), CT 8 (n = 21), CT 14 (n = 22), and CT 20 (n = 22). All groups preferred vanilla. There is a slight but significant difference in the data between CT 2 and CT 8 (P < 0.05, Kruskal–Wallis one-way ANOVA with Dunn's post hoc analysis) that is largely due to a particularly strong preference for vanilla of the animals tested at CT 2. (B) Change in PPI between the pre- and posttraining tests. There was a significant dependence on CT (Kruskal–Wallis one-way ANOVA, P < 0.001). Significant differences between groups (P < 0.05, Dunn's post hoc analysis) are indicated by differences in letters at the top of each bar. (C) Mean PPI when animals were retested 30 min after training. (D) Results of testing at CT 2 and CT 14 before training and 5 min after training. At CT 2, training had no effect, whereas at CT 14, there is a large shift in the PPI (P < 0.001).