Serotonin receptor activity is necessary for olfactory learning and memory in Drosophila melanogaster

Abstract

Learning and memory in the fruit fly, Drosophila melanogaster, is a complex behavior with many parallels to mammalian learning and memory. Although many neurotransmitters including acetylcholine, dopamine, glutamate, and GABA have previously been demonstrated to be involved in aversive olfactory learning and memory, the role of serotonin has not been well defined. Here, we present the first evidence of the involvement of individual serotonin receptors in olfactory learning and memory in the fly. We initially followed a pharmacological approach, utilizing serotonin receptor agonists and antagonists to demonstrate that all serotonin receptor families present in the fly are necessary for short-term learning and memory. Isobolographic analysis utilizing combinations of drugs revealed functional interactions are occurring between 5-HT(1A)-like and 5-HT(2), and 5-HT(2) and 5-HT(7) receptor circuits in mediating short-term learning and memory. Examination of long-term memory suggests that 5-HT(1A)-like receptors are necessary for consolidation and important for recall, 5-HT(2) receptors are important for consolidation and recall, and 5-HT(7) receptors are involved in all three phases. Importantly, we have validated our pharmacological results with genetic experiments and showed that hypomorph strains for 5-HT(2)Dro and 5-HT(1B)Dro receptors, as well as knockdown of 5-HT(7)Dro mRNA, significantly impair performance in short-term memory. Our data highlight the importance of the serotonin system and individual serotonin receptors to influence olfactory learning and memory in the fly, and position the fly as a model system to study the role of serotonin in cognitive processes relevant to mammalian CNS function.

Figure 1. 5-HT Receptor Agents Attenuate STM Performance in a Concentration-Dependent Manner

To determine the effects of 5-HT receptor agents on STM performance levels, 1–3 day old wild-type Canton-S flies were maintained on a solution of 1% agarose and 10% sucrose in the presence or absence of drug for 2 days prior to training. Flies demonstrated a concentration-dependent decrease in performance indices in the presence of all 5-HT receptor agents tested (n=8). The potency of the 5-HT_1A agonist to disrupt memory may reflect the high levels of expression of 5-HT_1ADro and 5-HT_1BDro receptors in the mushroom bodies of the adult brain.

Figure 2. 5-HT receptor mutants and knockdown of gene expression display decreased STM function

The 5-HT_1BDro and 5-HT₂Dro receptor hypormorph insertions in the wild-type CS background result in significant impairment of STM compared to CS. There were no significant differences observed among 5-HT₇Dro receptor parental Gal4 and RNAi driver lines. F1 progeny had significant impairment of STM. All flies displayed normal olfactory avoidance and shock reactivity. (n=8 for each genotype; *p<0.05, Student’s-t test for A and B, two-way ANOVA with Bonferroni post hoc test for multiple comparison for C).

Figure 3. Isobolar Analysis of Significant Receptor/Drug Interactions

Isobolographic analysis determined the nature of interactions between two receptors/drugs and their effects on STM performance. The IC₅₀ performance levels for a given drug at one receptor and that for another drug at a different receptor were compared to the effects on STM of a combination of the drugs. Experimental drug combination IC₅₀ values that were found to lie on the theoretical IC₅₀ isobole were considered additive; values that were above and below the isobole were considered superadditive and subadditive, respectively. Only isoboles for statistically significant interactions are shown.

Figure 4. 5-HT receptors differentially modulate acquisition, consolidation and retrieval of LTM

Based upon the STM results, 0.03mM U92106, 0.3mM DOI, and 0.3mM SB258719 was administered to 3–5 day old flies according to the above drug treatment protocol. The 5-HT_1A agonist had a significant effect on LTM consolidation, and also seems to play a role in retrieval. The 5-HT₂ agonist appears to play a role in both LTM consolidation and its retrieval. The 5-HT₇ antagonist appears to be important in all three aspects of LTM, and is equally important in consolidation and retrieval. (n=4 trials/treatment;*p<0.05 compared with control, one-way ANOVA with Dunnett’s post test for multiple comparison).