DNA methylation mediates the discriminatory power of associative long-term memory in honeybees

Abstract

Memory is created by several interlinked processes in the brain, some of which require long-term gene regulation. Epigenetic mechanisms are likely candidates for regulating memory-related genes. Among these, DNA methylation is known to be a long lasting genomic mark and may be involved in the establishment of long-term memory. Here we demonstrate that DNA methyltransferases, which induce and maintain DNA methylation, are involved in a particular aspect of associative long-term memory formation in honeybees, but are not required for short-term memory formation. While long-term memory strength itself was not affected by blocking DNA methyltransferases, odor specificity of the memory (memory discriminatory power) was. Conversely, perceptual discriminatory power was normal. These results suggest that different genetic pathways are involved in mediating the strength and discriminatory power of associative odor memories and provide, to our knowledge, the first indication that DNA methyltransferases are involved in stimulus-specific associative long-term memory formation.

Figure 1. DNA methyltransferase inhibition reduces the discriminatory power of olfactory long-term memory retrieval.

Four groups of bees (columns) were trained using appetitive olfactory conditioning and evaluated for acquisition (first row), memory retrieval (second row) and discriminatory power (third row). Each group was divided into two subgroups, one was treated with zebularine and the other was treated with the solvent DMF. Learning was quantified through observations of the conditioned-stimulus (CS) evoked proboscis extension response (PER). The sequence of CS and new odorant was balanced. Discriminatory power of the memory retrieval was quantified as the difference between a bees' response to CS and new odorant (1, PER to the CS only; 0, PER to CS and new, -1, PER to new only). (A ii-iv) Zebularine- and solvent-treated bees learned equally well during 3-trial training (p>0.18, Fisher's exact test). In the memory retrieval test (B i) 30 minutes after 1-trial training and (B ii) 30 minutes, (B iii) 1 day and (B iv) 3 days after 3-trial training bees responded more to the CS than to the new odorant (p<0.001, McNemar test). Memory retrieval did not differ in discriminatory power between zebularine- and solvent-treated bees when tested 30 minutes after (C i) 1-trial training (zebularine group: n = 44; solvent group: n = 46; p = 0.56, Welch's two sample t-test) or (C ii) 3-trial training (zebularine group: n = 49; solvent group: n = 51; p = 0.58, Welch's two sample t-test). Memory retrieval was less odor specific in zebularine-treated bees than in solvent-treated bees when tested (C iii) 1 day (zebularine group: n = 74; solvent group: n = 65; p = 0.008, Welch's two sample t-test) or (C iv) 3 days (zebularine group: n = 73; solvent group: n = 55; p = 0.03, Welch's two sample t-test) after 3-trial training.

Figure 2. DMF alone does not affect learning and memory retrieval one day after training.

(A) DMF-treated and untreated bees showed no significant difference in learning during 3-trial training (p>0.1, Fisher's exact test). n indicates the number of bees. (B) In the memory retrieval test, 1 day after training, bees responded more to the CS (1-hexanol) than to the new odorant (1-nonanol) (solvent group: n = 44; untreated group: n = 48; p = 0.038 and p = 0.004, McNemar test). The sequence of CS and new odor was balanced. (C) Memory retrieval did not differ in discriminatory power between DMF treated and untreated bees (p = 0.68, Welch's two sample t-test).

Figure 3. Zebularine treatment does not reduce the perceptual discriminatory power.

Bees were treated with zebularine or with the solvent DMF 24 hours before conditioning. (A) There was no significant difference in learning between zebularine- and solvent-treated bees during 3-trial training (p>0.68, Fisher's exact test). n indicates number of bees. (B) In the memory retrieval test, 1 day after training, bees responded more to the CS (1-hexanol) than to the new odorants 1-nonanol or citral (zebularine group: n = 48; solvent group: n = 48; p<0.001, McNemar test). The sequence of CS and 1-nonanol was balanced; citral was always presented as the last odor. (C) Memory retrieval did not differ in discriminatory power between zebularine- and solvent-treated bees (1-nonanol p = 0.59; citral p = 1, Welch's t-test).

Figure 4. Zebularine treatment does not reduce bee survival.

Bees were either treated with zebularine, DMF or were left untreated, and were kept for 72 hours. Every 24 hours the number of living bees was counted. A total of 49 bees were tested per group. The experiment was repeated seven times with 4–10 bees per replicate over a period of six months. The survival rates did not differ between the 3 groups (generalized linear mixed model, factor time: p<0.001, factor DMF treatment: p = 0.887, factor zebularine treatment: p = 0.253).